JP2016128405A - Ca6 antigen-specific cytotoxic conjugate and method of using the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a CA6 antigen-specific cytotoxic conjugate on a Muc1 mucin receptor, and to provide a method of using the same.SOLUTION: The invention discloses a cytotoxic conjugate comprising a cell binding agent and a cytotoxic agent. The invention also discloses therapeutic compositions comprising the conjugate, methods for using the conjugates in the inhibition of cell growth and the treatment of disease, and a kit comprising the cytotoxic conjugate. The cell binding agent is a monoclonal antibody and epitope-binding fragments thereof that recognize and bind the CA6 glycotope. The cytotoxic agent is a compound selected from a maytansinoid compound illustrated by a formula (II) or a taxoid compound, a CC-1065 compound, and the like.SELECTED DRAWING: None

Description

Related applications
[01] This application claims priority from US Provisional Patent Application 60 / 488,447 (filed July 21, 2003).

Field of Invention
[02] The present invention relates to murine anti-CA6 glycotope monoclonal antibodies, and humanized or resurfaced versions thereof. The invention also relates to epitope-binding fragments of the anti-CA6 glycotope monoclonal antibodies described above, and humanized or resurfaced versions of the epitope-binding fragments of the anti-CA6 glycotope monoclonal antibodies described above.

  [03] The present invention further includes a cytotoxic conjugate comprising a cell binding agent and a cytotoxic agent, a therapeutic composition comprising the conjugate, a method of using the conjugate for inhibiting cell proliferation and treating a disease, and It relates to a kit comprising a cytotoxic conjugate. The cell binding agent is in particular a monoclonal antibody that recognizes and binds to the CA6 glycotope or an epitope-binding fragment thereof, or a humanized or resurfaced version thereof.

Background of the Invention
[04] There have been many attempts to develop anti-cancer therapies that specifically destroy target cancer cells without harming surrounding non-cancerous cells and tissues. Such therapeutic agents have the potential to greatly improve cancer therapy in human patients.

[05] One promising method was to link cell toxins with cell binding agents such as monoclonal antibodies (Sela et al., Immunoconjugates 189-216 (C. Vogel, 1987)).
Ghose et al., Targeted Drugs 1-22 (E. Goldberg, 1983); Diener et al., Antibody mediated delivery systems 1-23 (J. Rodwell, 1988); Pietersz et al., Antibody mediated delivery systems 25-53 (J. Rodwell, 1988); Bumol et al., Antibody mediated delivery systems 55-79 (J. Rodwell, 1988)). Depending on the choice of cell binding agent, these cytotoxic conjugates can be designed to recognize and bind only certain types of cancer cells based on the expression profile of the molecules expressed on the surface of the cancer cells.

[06] Such cytotoxic conjugates include cytotoxics linked to various murine monoclonal antibodies, such as methotrexate, daunorubicin, doxorubicin, vincristine, vinblastine, Melphalan, mitomycin C, and chlorambucil have been used. In some cases, for example, drug molecules were linked to antibody molecules by the following intervening carrier molecules: serum albumin (Garnet et al., 46 Cancer Res. 2407-2412 (1986); Ohkawa et al., 23 Cancer Immunol. Immunolther 81-86 (1986); Endo et al., 47 Cancer Res. 1076-1080 (1980)), dextran (Hurwitz
et al., 2 Appl. Biochem. 25-35 (1980); Manabi et al., 34 Biochem. Pharmacol. 289-291 (1985); Dillman et al., 46 Cancer Res. 4886-4891 (1986); et al., 85
Proc. Natl. Acad. Sci. 8276-8280 (1988)), or polyglutamic acid (Tsukada et al., 73 J. Natl. Canc. Inst. 721-729 (1984); Kato et al., 27 J. Med Chem. 1602-1607 (1984); Tsukada et al., 52 Br. J. Cancer 111-116 (1985)).

[07] An example of a specific conjugate that has shown some promise is a conjugate of the C242 antibody formed against the antigen CanAg expressed in colorectal and pancreatic tumors and the maytansine derivative DM1. (Liu et al., Proc. Natl. Acad. Sci. USA, 93: 8618-8623 (1996)). In vitro evaluation of this conjugate shows that its binding affinity for CanAg expressed on the cell surface is as high as an apparent _Kd value of 3 × 10 ⁻¹¹ M and its cytotoxic potency against CanAg positive cells is IC ₅₀ 6 × 10 6. He pointed out that it was high at ^-11M . This cytotoxicity was blocked by excess unbound antibody and the sensitivity of antigen negative cells to this conjugate was more than 100 times lower, so the cytotoxicity was antigen dependent. Other examples of antibody-DM1 conjugates with both high affinity for each target cell and high antigen selective cytotoxicity include conjugates with: huN901, a humanized version antibody against human CD56; huMy9-6, a humanized version antibody against human CD33; huN242, a humanized version antibody against the CanAg Mucl epitope; huJ591, a deimmunized antibody against PSMA; trastuzumab, a humanized antibody against Her2 / neu; Humanized antibody against CD44v6.

[08] The development of other cytotoxic conjugates that specifically recognize specific types of cancer cells may be important in continuing to improve the methods used to treat cancer patients.
[09] Therefore, the present invention relates to the development of an antibody that recognizes and binds to a molecule / receptor expressed on the surface of a cancer cell, and a molecule expressed on the surface of a cancer cell, including a cell binding agent such as an antibody and a cytotoxic substance. / To develop new cytotoxic conjugates that specifically target receptors.

  [10] More specifically, the present invention recognizes the novel CA6 sialoglycotope characterization on the Muc1 mucin receptor expressed by cancer cells, and the novel CA6 sialoglycotope of Muc1 mucin. The aim is to provide an antibody, preferably a humanized antibody, that can be used in connection with a cytotoxic agent to inhibit the growth of CA6 glycotope expressing cells.

Summary of the Invention
[11] The present invention includes an antibody or epitope-binding fragment thereof that specifically recognizes and binds to a novel CA6 sialoglycotope of the Mucl mucin receptor. In other embodiments, the invention includes a humanized antibody or epitope-binding fragment thereof that recognizes a novel CA6 sialoglycotope (“CA6 glycotope”) of the Mucl mucin receptor.

[12] In another preferred embodiment, the present invention provides a murine anti-CA6 monoclonal antibody DS6 ("DS6 antibody"), and a resurfacing or humanized version of the DS6 antibody, wherein the L chain of the antibody or epitope-binding fragment thereof and In both heavy chains, antibodies are substituted with residues exposed on the surface that are more similar to the surface of a known human antibody. The humanized antibodies and epitope-binding fragments thereof of the present invention have been improved in that their immunogenicity is much less (or completely non-immunogenic) than the fully murine version in the human subject to which they are administered. Has characteristics. Accordingly, the humanized antibodies and epitope-binding fragments thereof of the present invention specifically recognize a novel sialoglycotope on the Mucl mucin receptor, namely the CA6 glycotope, but are not immunogenic to humans. These humanized antibodies and epitope-binding fragments thereof are specific for antigen-expressing cells by conjugating them to drugs, such as maytansinoids, and targeting the drugs to the Mucl CA6 sialoglycotope Prodrugs with high cytotoxicity can be formed. Accordingly, cytotoxic conjugates comprising such antibodies and small molecule highly toxic drugs (eg, maytansinoids, taxanes, and CC-1065 analogs) can be used to treat tumors such as breast and ovarian tumors. Can be used as a remedy for.

  [13] The humanized version DS6 antibody of the present invention can be obtained by using the amino acid sequences of the variable regions of both the L chain and H chain, the DNA sequences of the genes of the L chain and H chain variable regions, CDR identification, surface amino acids thereof Is fully elucidated herein with respect to the identification of and the disclosure of means for expressing them in recombinant form.

  [14] In one embodiment, the amino acid sequence represented by SEQ ID NOs: 1-3:

H chain containing CDRs having the amino acid sequence represented by SEQ ID NOs: 4-6:

A humanized DS6 antibody or epitope-binding fragment thereof having a light chain comprising a CDR having
[15] Amino acid sequence represented by SEQ ID NO: 7 or SEQ ID NO: 8:

Also provided are humanized DS6 antibodies and epitope-binding fragments thereof having a light chain variable region having an amino acid sequence with at least 90% sequence identity to.
[16] Similarly, the amino acid sequence represented by SEQ ID NO: 9, SEQ ID NO: 10, or SEQ ID NO: 11:

Humanized DS6 antibodies and epitope-binding fragments thereof having heavy chain variable regions with amino acid sequences with at least 90% sequence identity to are provided.
[17] In another embodiment, SEQ ID NO: 8:

A humanized DS6 antibody and an epitope-binding fragment thereof having a humanized or resurfaced light chain variable region having an amino acid sequence corresponding to
[18] Similarly, SEQ ID NO: 10 or SEQ ID NO: 11 respectively

A humanized DS6 antibody and an epitope-binding fragment thereof having a humanized or resurfaced heavy chain variable region having an amino acid sequence corresponding to
[19] The humanized DS6 antibodies and epitope-binding fragments thereof of the present invention also include substitutions at one or more positions of the light chain and / or heavy chain amino acid residues (as defined by the asterisk residues in Table 1). be able to. These positions represent murine surface framework residues that need to be exchanged for human residues and that are within 5 cm of the CDRs. For example, the first amino acid residue Q of the murine sequence (SEQ ID NO: 7) was replaced with E (SEQ ID NO: 8) to humanize the antibody. However, since this residue is close to the CDR, a back mutation to murine residue Q may be required to maintain antibody affinity.

  [20] This is further shown in Table 2. This table shows muDS6 variable region surface residues aligned with the three most highly homologous human variable region surface residues. The amino acid residues in Table 1 correspond to the amino acid residues underlined in Table 2.

[21] The present invention further provides a cytotoxic conjugate comprising (1) a cell binding agent that recognizes and binds to a CA6 glycotope, and (2) a cytotoxic substance. In the cytotoxic conjugate, the cell binding agent has a high affinity for the CA6 glycotope, and the cytotoxic agent has a high cytotoxicity for the cell expressing the CA6 glycotope. Therefore, the cytotoxic conjugate of the present invention is effective. Forms a cell killing agent.

[22] In a preferred embodiment, the cell binding agent is an anti-CA6 antibody or epitope-binding fragment thereof, more preferably a humanized anti-CA6 antibody or epitope-binding fragment thereof, and the cytotoxic agent is directly or cleavable or non-cleavable. It is covalently attached to the antibody or epitope-binding fragment thereof via a sex linker. In a more preferred embodiment, the cell binding agent is a humanized DS6 antibody or an epitope binding fragment thereof and the cytotoxic agent is a taxol, maytansinoid, CC-1065 or C
C-1065 analog.

[23] In a preferred embodiment of the present invention, the cell binding agent is a humanized anti-CA6 antibody and the cytotoxic agent is a cytotoxin such as maytansinoids or taxanes.
[24] More preferably, the cell binding agent is a humanized anti-CA6 antibody DS6 and the cytotoxic agent is a maytansine compound, such as DM1 or DM4.

  [25] The present invention also includes a method of inhibiting the growth of CA6 glycotope-expressing cells. In a preferred embodiment, the method of inhibiting the growth of CA6 glycotope expressing cells is performed in vivo to kill the cells, although in vitro and ex vivo applications are also included.

[26] The present invention also provides a therapeutic composition comprising a cytotoxic conjugate and a pharmaceutically acceptable carrier or excipient.
[27] The present invention further includes a method of treating a subject with cancer using the therapeutic composition of the present invention. In preferred embodiments, the cytotoxic conjugate comprises an anti-CA6 antibody and a cytotoxic agent. In a more preferred embodiment, the cytotoxic conjugate comprises a humanized DS6 antibody-DM1 conjugate, a humanized DS6 antibody-DM4 conjugate, or a humanized DS6 antibody-taxane conjugate, wherein the conjugate is a pharmaceutically acceptable carrier. Or administered with excipients.

  [28] The present invention also includes a kit comprising an anti-CA6 antibody-cytotoxic agent conjugate and instructions for use. In a preferred embodiment, the anti-CA6 antibody is a humanized DS6 antibody, the cytotoxic agent is a maytansine compound such as DM1 or DM4, or taxanes, and the indication is directed to treating the conjugate of the invention in treating a subject with cancer. Instructions for use. The kit also contains the components necessary to prepare a pharmaceutically acceptable formulation, such as a diluent if the conjugate is in lyophilized or concentrated form, and the components necessary to administer the formulation. Including.

  [29] The present invention also includes derivatives of antibodies that specifically recognize and bind to the CA6 glycotope. In preferred embodiments, antibody derivatives are prepared by resurfacing or humanizing antibodies that bind CA6 glycotopes, which derivatives have reduced immunogenicity to the host.

  [30] The present invention further provides a humanized antibody or fragment thereof that is further labeled for use in research or diagnostic applications. In preferred embodiments, the label is a radioactive label, fluorophore, chromophore, contrast agent or metal ion.

  [31] A diagnostic method is also provided that includes administering a labeled humanized antibody or epitope-binding fragment thereof to a subject suspected of having cancer, and measuring or monitoring the distribution of the label in the subject's body.

[32] The present invention also provides a method of treating a subject with cancer by administering a humanized antibody conjugate of the present invention alone or in combination with other cytotoxic agents or therapeutic agents. The cancer is, for example, breast cancer (breast cancer), colon cancer, ovarian cancer, endometrial cancer, osteosarcoma, cervical cancer, prostate cancer, lung cancer, synovial cancer, pancreatic cancer, sarcoma or cancer expressing CA6, or CA6 There may be one or more types of other cancers that mainly express glycotopes that have not yet been determined.

  [33] Unless otherwise noted, all references and patents cited herein are hereby incorporated by reference.

FIG. 1 shows the results of tests performed to determine the ability of DS6 antibodies to bind to the surface of specific cancer cell lines. The fluorescence of cell lines incubated with DS6 primary antibody and FITC-conjugated anti-mouse IgG (H + L) secondary antibody was measured by flow cytometry. The DS6 antibody bound Caov-3 (FIG. 1A) and T-47D (FIG. 1B) cells with an apparent Kd of 1.848 nM and 2.586 nM, respectively. Antigen negative cell lines SK-OV-3 (FIG. 1C) and Colo205 (FIG. 1D) did not show antigen-specific binding. FIG. 2 shows the results of a dot blot analysis of epitope expression. Caov-3 (Figures 2A and 2B), SKMEL28 (Figure 2C) and Colo205 (Figure 2D) cell lysates are individually spotted on a nitrocellulose membrane and then individually with pronase, proteinase K, neuraminidase or periodate Incubated. The membrane was then immunoblotted with DS6 antibody (FIG. 2A), CM1 antibody (FIG. 2B), R24 antibody (FIG. 2C), or C242 antibody (FIG. 2D). FIG. 3 shows the results of dot blot analysis of DS6 antigen expression. Caov-3 cell lysates were individually spotted on PVDF membranes and then incubated in the presence of trifluoromethanesulfonic acid (TFMSA). The membrane was then immunoblotted with CM1 antibody (1 and 2) or DS6 antibody (3 and 4). FIG. 4 shows the results of glycotope analysis of the DS6 antigen. Caov-3 cell lysate pretreated with N-glycanase ("N-gly"), O-glycanase ("O-gly"), and / or sialidase ("S") is spotted onto nitrocellulose, then Immunoblotting was performed with DS6 antibody or CM1 antibody (Muc1 VNTR). FIG. 5 shows the results of Western blot analysis of the DS6 antigen. Cell lysates were immunoprecipitated ("IP") and immunoblotted with DS6 antibody. The antigen corresponds to the> 250 kDa protein band found in antigen positive Caov-3 (FIGS. 5A and 5B) and T47D (FIG. 5C) cells. Antigen negative SK-OV-3 (FIG. 5D) and Colo205 (FIG. 5E) cell lines do not show this band. After immunoprecipitation, the protein G band of the Caov-3 cell lysate was incubated with (FIG. 5A) neuraminidase (“N”) or (FIG. 5B) periodic acid (“PA”). Antibody (“α”), pre-IP (“Lys”) and post-IP flow-through (“FT”) lysate controls were run on the same gel. Caov-3 immunoprecipitates were similarly incubated with N-glycanase (“N-gly”), O-glycanase (“O-gly”), and / or sialidase (“S”) (see FIG. 5F) and blotted Instead were tested with biotinylated-DS6 and streptavidin-HRP. FIG. 5 shows the results of Western blot analysis of the DS6 antigen. Cell lysates were immunoprecipitated ("IP") and immunoblotted with DS6 antibody. The antigen corresponds to the> 250 kDa protein band found in antigen positive Caov-3 (FIGS. 5A and 5B) and T47D (FIG. 5C) cells. Antigen negative SK-OV-3 (FIG. 5D) and Colo205 (FIG. 5E) cell lines do not show this band. After immunoprecipitation, the protein G band of the Caov-3 cell lysate was incubated with (FIG. 5A) neuraminidase (“N”) or (FIG. 5B) periodic acid (“PA”). Antibody (“α”), pre-IP (“Lys”) and post-IP flow-through (“FT”) lysate controls were run on the same gel. Caov-3 immunoprecipitates were similarly incubated with N-glycanase (“N-gly”), O-glycanase (“O-gly”), and / or sialidase (“S”) (see FIG. 5F) and blotted Instead were tested with biotinylated-DS6 and streptavidin-HRP. FIG. 6 shows the results of immunoprecipitation and / or immunoblotting of DS6 and CM1 antibodies on Caov-3 (FIG. 6A) and HeLa (FIG. 6B) cell lysates. The overlapping CM1 and DS6 Western blot signals indicate that the DS6 antigen is on the Muc1 protein. In HeLa cell lysates, Muc1 doublets result from Muc1 expression governed by distinct alleles with different numbers of tandem repeats. FIG. 7 shows the DS6 antibody sandwich ELISA design (FIG. 7A) and standard curve (FIG. 7B). Standard curves were generated using known concentrations of commercial CA15-3 standards (1 CA15-3 units = 1 DS6 units). FIG. 8 shows a quantitative ELISA standard curve. Standard curve of detection antibody (streptavidin-HRP / biotin-DS6) signal (FIG. 8C) captured by plate-bound goat anti-mouse IgG (FIG. 8A) or directly bound to ELISA plate (FIG. 8B), known Made with a concentration of biotin-DS6. FIG. 9 shows the cDNA and amino acid sequences of the murine DS6 antibody L chain (FIG. 9A) and H chain (FIG. 9B) variable regions. Three CDRs in each sequence are underlined (Kabat definition). FIG. 10 shows the light chain (FIG. 10A) and H chain (FIG. 10B) CDRs of murine DS6 antibody as determined by Kabat definition. AbM modeling software makes a slight difference in the definition for heavy chain CDRs (FIG. 10C). FIG. 11 shows the murine DS6 antibody L chain (“muDS6LC”) (SEQ ID NO: 7 residues 1-95) and H chain (“muDS6HC”) (SEQ ID NO: 9 residues 1-98). The amino acid sequence is IgV? Shown are aligned with germline sequences of the ap4 (SEQ ID NO: 23) and IgVh J558.41 (SEQ ID NO: 24) genes. Gray indicates sequence differences. FIG. 12 shows 10 L and H chains that are most homologous to murine DS6 (muDS6) L chain (“muDS6LC”) and H chain (“muDS6HC”) sequences and have resolved structure files in the Brookhaven database. The antibody sequence is shown. The sequences were arranged in order from highest to lowest homology. FIG. 13 shows surface accessibility data and calculated values for estimating which framework residues of the murine DS6 antibody light chain variable region are surface accessible. Mark an average surface accessibility 25% to 35% of the positions ^(* ?? ^*) and were subjected to analysis of the second round. DS6 antibody L chain variable region (FIG. 13A) and H chain variable region (FIG. 13B). FIG. 13 shows surface accessibility data and calculated values for estimating which framework residues of the murine DS6 antibody light chain variable region are surface accessible. Mark an average surface accessibility 25% to 35% of the positions ^(* ?? ^*) and were subjected to analysis of the second round. DS6 antibody L chain variable region (FIG. 13A) and H chain variable region (FIG. 13B). FIG. 14 shows the prDS6v1.0 mammalian expression plasmid map. This plasmid was used to construct and express recombinant chimeric and humanized DS6 antibodies. FIG. 15 shows the amino acid sequences of the variable domains of the murine DS6 antibody (“muDS6”) and humanized DS6 antibody (“huDS6”) (v1.0 and v1.2) (FIG. 15A) and H chain (FIG. 15B). Indicates. FIG. 16 shows the cDNA and amino acid sequences of the light chain variable region of the humanized DS6 antibody (“huDS6”) (v1.0 and v1.2). FIG. 17 shows the cDNA and amino acid sequences of the heavy chain variable region of humanized DS6 antibody (“huDS6”) v1.0 (FIG. 17A) and v1.2 (FIG. 17B). FIG. 18 shows flow cytometry binding curves for muDS6 and huDS6 clones from assays performed on WISH cells. (FIG. 18A) The binding affinities of the chimeric, v1.0, and v1.2 human DS6 clones (Kd = 3.15, 3.71 and 4.20 nM, respectively) (FIG. 18B) of the naked and biotinylated murine DS6. It was equivalent to the binding affinity (Kd = 1.93 and 2.80 nM, respectively). FIG. 19 shows the results of a competitive binding assay of huDS6 antibody and muDS6. (FIG. 19A) WISH cells were incubated with biotin-muDS6 and streptavidin-DTAF to obtain a binding curve with an apparent Kd of 6.76 nM. (FIG. 19B) Various concentrations of naked muDS6, huDS6 v1.0 and v1.2 were conjugated with 2 nM biotin-muDS6 and then conjugated with streptavidin-DTAF. FIG. 20 shows the results of measuring the binding affinity of unconjugated DS6 antibody compared to DS6 antibody-DM1 conjugate. These results demonstrate that DM1 conjugation does not deleteriously affect the binding affinity of the antibody. The apparent Kd (3.902 nM) (“DS6-DM1”) of the DS6 antibody-DM1 conjugate was slightly larger than that of the naked antibody (2.020 nM) (“DS6”). FIG. 21 shows the results of an indirect cell viability assay using DS6 antibody in the presence or absence of anti-murine IgG (H + L) DM1 conjugate (2 ° Ab-DM1). Antigen-positive Caov-3 cells died in a DS6 antibody-dependent manner (IC ₅₀ = 424.9 pM) only in the presence of the secondary conjugate (“DS6 + 2 ° Ab-DM1”). FIG. 22 shows the results of complement dependent cytotoxicity (CDC) of DS6 antibody and humanized DS6 antibody. These results demonstrate that there is no CDC-mediated effect of DS6 or humanized DS6 antibodies (v1.0 and v1.2) on HPAC cells (FIG. 22A) and ZR-75-1 cells (FIG. 22B). did. FIG. 23 shows the results of an in vitro cytotoxicity assay of DS6 antibody-DM1 conjugate versus free maytansine. In colony formation assays, ovarian cancer (FIG. 23A), breast cancer (FIG. 23B), cervical cancer (FIG. 23C) and pancreatic cancer (FIG. 23D) cell lines positive for the DS6 antigen were transferred to the DS6 antibody-DM1 conjugate. Were tested for cytotoxicity of the continuous exposure (left panel). These cell lines were similarly tested for maytansine sensitivity by 72 hours exposure to free maytansine (right panel). The ovarian cancer cell lines tested were OVCAR5, TOV-21G, Caov-4 and Caov-3. The breast cancer cell lines tested were T47D, BT-20 and BT-483. The cervical cancer cell lines tested were KB, HeLa and WISH. The pancreatic cancer cell lines tested were HPAC, Hs766T and HPAF-II. FIG. 23 shows the results of an in vitro cytotoxicity assay of DS6 antibody-DM1 conjugate versus free maytansine. In colony formation assays, ovarian cancer (FIG. 23A), breast cancer (FIG. 23B), cervical cancer (FIG. 23C) and pancreatic cancer (FIG. 23D) cell lines positive for the DS6 antigen were transferred to the DS6 antibody-DM1 conjugate. Were tested for cytotoxicity of the continuous exposure (left panel). These cell lines were similarly tested for maytansine sensitivity by 72 hours exposure to free maytansine (right panel). The ovarian cancer cell lines tested were OVCAR5, TOV-21G, Caov-4 and Caov-3. The breast cancer cell lines tested were T47D, BT-20 and BT-483. The cervical cancer cell lines tested were KB, HeLa and WISH. The pancreatic cancer cell lines tested were HPAC, Hs766T and HPAF-II. FIG. 23 shows the results of an in vitro cytotoxicity assay of DS6 antibody-DM1 conjugate versus free maytansine. In colony formation assays, ovarian cancer (FIG. 23A), breast cancer (FIG. 23B), cervical cancer (FIG. 23C) and pancreatic cancer (FIG. 23D) cell lines positive for the DS6 antigen were transferred to the DS6 antibody-DM1 conjugate. Were tested for cytotoxicity of the continuous exposure (left panel). These cell lines were similarly tested for maytansine sensitivity by 72 hours exposure to free maytansine (right panel). The ovarian cancer cell lines tested were OVCAR5, TOV-21G, Caov-4 and Caov-3. The breast cancer cell lines tested were T47D, BT-20 and BT-483. The cervical cancer cell lines tested were KB, HeLa and WISH. The pancreatic cancer cell lines tested were HPAC, Hs766T and HPAF-II. FIG. 23 shows the results of an in vitro cytotoxicity assay of DS6 antibody-DM1 conjugate versus free maytansine. In colony formation assays, ovarian cancer (FIG. 23A), breast cancer (FIG. 23B), cervical cancer (FIG. 23C) and pancreatic cancer (FIG. 23D) cell lines positive for the DS6 antigen were transferred to the DS6 antibody-DM1 conjugate. Were tested for cytotoxicity of the continuous exposure (left panel). These cell lines were similarly tested for maytansine sensitivity by 72 hours exposure to free maytansine (right panel). The ovarian cancer cell lines tested were OVCAR5, TOV-21G, Caov-4 and Caov-3. The breast cancer cell lines tested were T47D, BT-20 and BT-483. The cervical cancer cell lines tested were KB, HeLa and WISH. The pancreatic cancer cell lines tested were HPAC, Hs766T and HPAF-II. FIG. 24 shows the results of an in vitro cytotoxicity assay of DS6 antibody-DM1 conjugate. In the MTT cell viability assay, human ovarian cancer (Figures 24A, 24B and 24C), breast cancer (Figures 24D and 24E), cervical cancer (Figures 24F and 24G) and pancreatic cancer (Figures 23H and 24I). ) Cell lines died dependent on DS6 antibody-DM1 conjugate. Naked DS6 had no detrimental effect on the growth of these cells. This indicates that DM1 conjugation is required for cytotoxicity. FIG. 24 shows the results of an in vitro cytotoxicity assay of DS6 antibody-DM1 conjugate. In the MTT cell viability assay, human ovarian cancer (Figures 24A, 24B and 24C), breast cancer (Figures 24D and 24E), cervical cancer (Figures 24F and 24G) and pancreatic cancer (Figures 23H and 24I). ) Cell lines died dependent on DS6 antibody-DM1 conjugate. Naked DS6 had no detrimental effect on the growth of these cells. This indicates that DM1 conjugation is required for cytotoxicity. FIG. 25A shows the results of an in vivo anti-tumor effect study of DS6 antibody-DM1 conjugates on established subcutaneous KB tumor xenografts. Tumor cells were inoculated on day 0 and the first treatment was performed on day 6. The immunoconjugate treatment was continued once a day for a total of 5 times. PBS control animals were euthanized when the tumor volume exceeded 1500 mm ³ . Conjugates were administered at a dose of DM1 150 or 225 μg / kg: corresponding to antibody concentrations of 5.7 and 8.5 mg / kg, respectively. During the test period, the body weight of the mice (FIG. 25B) was monitored. FIG. 26 shows the results of an anti-tumor effect test of DS6 antibody-DM1 conjugates on established subcutaneous tumor xenografts. OVCAR5 (FIGS. 26A and 26B), TOV-21G (FIGS. 26C and 26D), HPAC (FIGS. 26E and 26F), and HeLa (FIGS. 26G and 26H) cells were seeded on day 0, 6 and 13 Immunoconjugate treatment was performed on the day. PBS control animals were euthanized when tumor volume exceeded 1000 mm ³ . The conjugate was administered at a dose of DM1 600 μg / kg: this corresponds to an antibody concentration of 27.7 mg / kg. During the study period, tumor volume (FIGS. 26A, 26C, 26E, and 26G) and mouse body weight (FIGS. 26B, 26D, 26F, and 26H) were monitored. FIG. 26 shows the results of an anti-tumor effect test of DS6 antibody-DM1 conjugates on established subcutaneous tumor xenografts. OVCAR5 (FIGS. 26A and 26B), TOV-21G (FIGS. 26C and 26D), HPAC (FIGS. 26E and 26F), and HeLa (FIGS. 26G and 26H) cells were seeded on day 0, 6 and 13 Immunoconjugate treatment was performed on the day. PBS control animals were euthanized when tumor volume exceeded 1000 mm ³ . The conjugate was administered at a dose of DM1 600 μg / kg: this corresponds to an antibody concentration of 27.7 mg / kg. During the study period, tumor volume (FIGS. 26A, 26C, 26E, and 26G) and mouse body weight (FIGS. 26B, 26D, 26F, and 26H) were monitored. FIG. 26 shows the results of an anti-tumor effect test of DS6 antibody-DM1 conjugates on established subcutaneous tumor xenografts. OVCAR5 (FIGS. 26A and 26B), TOV-21G (FIGS. 26C and 26D), HPAC (FIGS. 26E and 26F), and HeLa (FIGS. 26G and 26H) cells were seeded on day 0, 6 and 13 Immunoconjugate treatment was performed on the day. PBS control animals were euthanized when tumor volume exceeded 1000 mm ³ . The conjugate was administered at a dose of DM1 600 μg / kg: this corresponds to an antibody concentration of 27.7 mg / kg. During the study period, tumor volume (FIGS. 26A, 26C, 26E, and 26G) and mouse body weight (FIGS. 26B, 26D, 26F, and 26H) were monitored. FIG. 26 shows the results of an anti-tumor effect test of DS6 antibody-DM1 conjugates on established subcutaneous tumor xenografts. OVCAR5 (FIGS. 26A and 26B), TOV-21G (FIGS. 26C and 26D), HPAC (FIGS. 26E and 26F), and HeLa (FIGS. 26G and 26H) cells were seeded on day 0, 6 and 13 Immunoconjugate treatment was performed on the day. PBS control animals were euthanized when tumor volume exceeded 1000 mm ³ . The conjugate was administered at a dose of DM1 600 μg / kg: this corresponds to an antibody concentration of 27.7 mg / kg. During the study period, tumor volume (FIGS. 26A, 26C, 26E, and 26G) and mouse body weight (FIGS. 26B, 26D, 26F, and 26H) were monitored. FIG. 27 shows the results of an in vivo efficacy study of DS6 antibody-DM1 conjugate on intraperitoneal OVCAR5 tumors. Tumor cells were injected intraperitoneally on day 0 and immunoconjugate treatment was performed on days 6 and 13. Animals were euthanized when weight loss exceeded 20%. FIG. 28 shows flow cytometry binding curves obtained from binding affinity studies of naked and taxane-conjugated DS6 antibodies on HeLa cells. Taxane (MM1-202) conjugation does not adversely affect the binding affinity of the antibody. The apparent Kd (1.24 nM) of the DS6-MM1-202 conjugate was slightly larger than that of the naked DS6 antibody (629 pM).

Detailed Description of the Invention
[62] The present invention specifically provides anti-CA6 monoclonal antibodies, anti-CA6 humanized antibodies, and anti-CA6 antibody fragments. The antibodies and antibody fragments of the present invention are each designed to specifically recognize and bind to the cell surface CA6 glycotope. Many human tumors are known to express CA6: 95% of serous ovarian cancer, 50% of endometrial ovarian cancer, 50% of cervical neoplasms, endometrial neoplasms 69% of vulvar neoplasms, 80% of vulvar neoplasms, 60% of breast cancers, 67% of pancreatic tumors, and 48% of urothelial tumors; however, normal human tissues are rarely expressed.

[63] Kearse et al., Int. J. Cancer 88 (6): 866-872 (2000) reported CA6
The protein in which the epitope was present was mistakenly identified as an 80 kDa protein with an N-linked carbohydrate containing the CA6 epitope; they used the hybridoma supernatant for identification. Subsequently, the inventors used purified DS6 to demonstrate that the CA6 epitope is on the O-linked carbohydrate of non-disulfide bond-glycoprotein greater than 250 kDa. Furthermore, this glycoprotein was identified as mucin Mucl. Since different Muc1 alleles have different numbers of tandem repeats in the variable number tandem repeat (VNTR) domain, cells often express two distinct Muc1 proteins of different sizes (Taylor-Papadimitriou, Biochim. Biophys. Acta 1455 (2-3): 301-13 (1999)). Due to differences in the number of VNTR domain repeats and glycosylation, the molecular weight of Mucl varies from cell line to cell line.

[64] The sensitivity of CA6 immunoreactivity to periodate indicates that CA6 is a carbohydrate epitope, or “glycotope”. Furthermore, the sensitivity of CA6 immunoreactivity to treatment with neuraminidase from Vibrio cholerae indicates that CA6 is a sialic acid-dependent glycotope, or “sialoglycotope”.

  [65] Details on characterization of CA6 are in Example 2 (see below). More details about CA6 can be found below:

  [66] The present invention also includes cytotoxic conjugates comprising two major components. The first component is a cell binding agent that recognizes and binds to the CA6 glycotope. The cell binding agent should recognize the CA6 sialoglycotopes on Muc1 with such high specificity that the cytotoxic conjugate recognizes and binds only the targeted cells. Due to the high degree of specificity, the conjugate can be targeted and act with little side effects resulting from non-specific binding.

  [67] In other embodiments, the cell binding agent of the present invention may also be of sufficient duration that the cytotoxic drug portion of the conjugate can act on the cell, and / or the conjugate is internalized into the cell. It recognizes the CA6 glycotope with a high enough affinity that the conjugate is in contact with the target cell for a sufficient period of time.

  [68] In a preferred embodiment, the cytotoxic conjugate comprises an anti-CA6 antibody as a cell binding agent, more preferably a murine DS6 anti-CA6 monoclonal antibody. In a more preferred embodiment, the cytotoxic conjugate comprises a humanized DS6 antibody or epitope binding fragment thereof. The DS6 antibody can recognize CA6 with a high degree of specificity and targets and directs cytotoxic agents to abnormal cells or tissues, such as cancer cells.

  [69] The second component of the cytotoxic conjugate of the present invention is a cytotoxic substance. In preferred embodiments, the cytotoxic agent is a taxol, maytansinoid, such as DM1 or DM4, CC-1065 or a CC-1065 analog. In a preferred embodiment, the cell binding agent of the present invention is covalently bound to the cytotoxic agent directly or via a cleavable or non-cleavable linker.

[70] Cell binding agents, cytotoxic substances, and linkers are described in detail below.
Cell binding agent
[71] The effectiveness of the compounds of the invention as therapeutic agents depends on careful selection of appropriate cell binding agents. Cell binding agents may be of any type now known or known in the future, including peptides and non-peptides. The cell binding agent may be any compound that can bind cells specifically or non-specifically. In general, these may be antibodies (especially monoclonal antibodies), lymphokines, hormones, growth factors, vitamins, nutrient transport molecules (eg transferrin), or any other cell-binding molecule or substance.

[72] More specific examples of binders that can be used include:
(A) a polyclonal antibody;
(B) a monoclonal antibody;
(C) Fragments of antibodies, such as Fab, Fab ′, and F (ab ′) ₂ , Fv (Parham, J. Immunol. 131: 2895-2902 (1983); Spring et al., J. Immunol. 113: 470 -478 (1974); Nisonoff et al., Arch. Biochem. Biophys. 89: 230-244 (1960));
(D) interferons (eg, α, β, γ);
(E) lymphokines such as IL-2, IL-3, IL-4, IL-6;
(F) hormones such as insulin, TRH (thyrotropin releasing hormone), MSH (melanocyte stimulating hormone), steroid hormones such as androgens and estrogens;
(G) Growth factors such as colony stimulating factors such as EGF, TGF-α, FGF, VEGF, G-CSF, M-CSF and GM-CSF (Burgess, Immunology Today 5: 155-158 (1984));
(H) transferrin (O'Keefe et al., J. Biol. Chem. 260: 932-937 (1985)); and (i) vitamins such as folic acids.

antibody
[73] The selection of an appropriate cell-binding agent depends on the individual cell population to be targeted, but an antibody is generally preferred and a monoclonal antibody is more preferred if an appropriate antibody can be obtained or prepared.

  [74] Monoclonal antibody methods can be used to prepare highly specific cell binding agents in the form of specific monoclonal antibodies. Particularly well-known methods in the art are the following: antigens of interest such as intact target cells, antigens isolated from target cells, whole virus bodies, attenuated whole virus bodies , And a monoclonal antibody produced by immunization with a viral protein, such as a viral coat protein. Sensitized human cells can also be used. Another method for producing monoclonal antibodies is the use of a phage library of scFv (single chain variable region), particularly human scFv (eg Griffiths et al., USP 5,885,793 and 5,969,108; McCafferty et al., WO 92/01047; see Liming et al., WO 99/06587).

[75] A typical antibody consists of two identical heavy chains and two identical light chains linked by disulfide bonds. The variable region is a part of the heavy and light chains of the antibody, the sequence of which varies between antibodies, and is the portion that coordinates in the binding and specificity of individual antibodies to the antigen. Variations are not necessarily distributed uniformly throughout the antibody variable region. Variations are generally concentrated in three segments called variable region complementarity determining regions (CDRs) or hypervariable regions in both the light and heavy chain variable regions. The variable part of the more highly conserved part is called the framework area. The variable regions of the H and L chains contain four framework regions, which often have a β-sheet structure, each framework region is linked by three CDRs, and the CDR contains a loop connecting the β-sheet structures. And optionally form part of the β sheet structure. The CDRs in each chain are held in close proximity by the framework regions and contribute to the formation of the antigen binding site of the antibody with the CDRs of the other chain (EAKabat et al., Sequences of
(Immunological Interest, 5th edition, 1991, NIH).

  [76] The constant region is a part of the H chain. It is not directly related to the binding of antibodies to antigens, but actually exhibits a variety of effector functions such as antibody involvement in antibody-dependent cytotoxicity.

[77] Monoclonal antibodies suitable for use in the present invention include murine DS6 monoclonal antibody (USP 6,596,503; ATCC deposit number PTA-4449).
Humanized or resurfaced DS6 antibody
[78] Preferably, a humanized anti-CA6 antibody is used as the cell binding agent of the present invention. A preferred embodiment of such a humanized antibody is a humanized DS6 antibody or an epitope-binding fragment thereof.

  [79] The goal of humanization is to reduce immunogenicity in order to introduce heterologous antibodies, such as murine antibodies, into humans while maintaining the full antigen binding affinity and specificity of the antibody.

  [80] Humanized antibodies can be prepared by several methods, including resurfacing and CDR grafting. The resurfacing method used in the present invention uses a combination of molecular modeling, statistical analysis and mutagenesis to change the non-CDR surface of the antibody variable region to resemble the surface of a known antibody in the target host.

[81] Strategies and methods for antibody resurfacing and other methods for reducing the immunogenicity of antibodies in different hosts are disclosed in USP 5,639,641 (Pedersen et al.), Which is incorporated herein in its entirety. To do. In summary, in a preferred method, (1) a positional alignment of antibody heavy and light chain variable region pools is created to determine a set of positions exposed on the surface of the heavy and light chain variable region frameworks, The alignment positions for all variable regions are at least about 98% identical; (2) for rodent antibodies (or fragments thereof), the set of amino acid residues exposed on the heavy and light chain variable region framework surfaces; Determine the group; (3) identify the set of amino acid residues exposed on the surface of the heavy and light chain variable region frameworks that have the closest identity to the surface exposed set of amino acid residues in the rodent (4) a set of amino acid residues exposed on the surface of the heavy chain and light chain variable region framework determined in step (2), and the complementarity determining region of the rodent antibody. Substitute a set of amino acid residues exposed on the surface of the heavy chain and light chain variable region frameworks determined in step (3) except for amino acid residues within 5 cm of any one of these residues. Thus (5) producing humanized rodent antibodies with binding specificity.

[82] Antibodies can be humanized in various other ways including: CDR grafting (EP 0 239 400; WO 91/09967; USP 5,530,101; and 5,585,089), veneering or resurfacing (EP 0 592 106; EP 519 596; Padlan EA, 1991, Molecular Immunology 28 (4/5): 489-498; Studnika GM et al., 1994, Protein Engineering 7 (6): 805-814; Roguska MA et al., 1994, PNAS 91: 969-973), and chain shuffling (USP 5,565,332). Human antibodies can be prepared by a variety of known methods including phage display methods. USP 4,444,887, 4,716,111, 5,545,806, and 5,814,318; and International Patent Application Publications WO 98/46645, WO 98/50433, WO 98/24893, WO 98/16654, WO
See also 96/34096, WO 96/33735, and WO 91/10741, the entire contents of which are incorporated herein by reference.

  [83] In a preferred embodiment, the present invention provides a humanized antibody or fragment thereof that recognizes a novel sialoglycotope (CA6 glycotope) on the Mucl mucin. In other embodiments, the humanized antibody or epitope-binding fragment thereof is further capable of inhibiting the growth of CA6 glycotope expressing cells.

  [84] In a more preferred embodiment, a humanized or resurfaced version of the DS6 antibody, wherein the exposed residues on the surface of both the light and heavy chains of the antibody or fragment thereof are further deposited on the surface of a known human antibody. Antibodies with substitutions are provided. The humanized DS6 antibody or epitope-binding fragment thereof of the present invention has improved properties. For example, the inventive humanized DS6 antibody or epitope-binding fragment thereof specifically recognizes a novel sialoglycotope (CA6 glycotope) on the Mucl mucin. More preferably, the humanized antibodies and epitope-binding fragments thereof of the present invention are further capable of inhibiting the growth of CA6 glycotope expressing cells. When humanized antibodies and epitope-binding fragments thereof of the present invention are conjugated to drugs, such as maytansinoids, they are specific for antigen-expressing cells by targeting the drug to the novel Mucl sialoglycotope CA6. Prodrugs with cytotoxicity can be formed. Cytotoxic conjugates comprising such antibodies and small molecule highly toxic drugs (eg maytansinoids, taxanes, and CC-1065 analogs) are used as therapeutic agents for the treatment of tumors such as breast and ovarian tumors. Can be used as

  [85] The humanized version DS6 antibody of the present invention can be obtained by using the amino acid sequences of both the heavy chain and light chain variable regions, the DNA sequences of both the light chain and heavy chain variable regions, CDR identification, The identification of surface amino acids as well as disclosure of means for expressing them in recombinant form is fully elucidated herein.

  [86] In one embodiment, the amino acid sequence represented by SEQ ID NOs: 1-3:

A humanized antibody or epitope-binding fragment thereof having a heavy chain comprising a CDR having
[87] When the heavy chain CDRs are determined by AbM modeling software, they are
ID NO: 20-22:

Is represented by
[88] In the same embodiment, the humanized antibody or epitope-binding fragment thereof is SEQ.
Amino acid sequence represented by ID NO: 4-6:

It has an L chain containing a CDR having
[89] Amino acid sequence represented by SEQ ID NO: 7 or SEQ ID NO: 8:

Also provided are humanized antibodies and epitope-binding fragments thereof having light chain variable regions having amino acid sequences with at least 90% sequence identity to.
[90] Similarly, an amino acid sequence represented by SEQ ID NO: 9, SEQ ID NO: 10, or SEQ ID NO: 11:

Humanized antibodies and epitope-binding fragments thereof having heavy chain variable regions having amino acid sequences with at least 90% sequence identity to.
[91] In other embodiments, SEQ ID NO: 8:

Humanized antibodies and epitope-binding fragments thereof having humanized or resurfaced light chain variable regions having amino acid sequences corresponding to are provided.
[92] Similarly, SEQ ID NO: 10, or SEQ ID NO: 11, respectively

Humanized antibodies and epitope-binding fragments thereof having humanized or resurfaced heavy chain variable regions having amino acid sequences corresponding to are provided.
[93] The humanized antibodies and epitope-binding fragments thereof of the present invention are of human surface amino acid residues adjacent to the CDRs of the light and / or heavy chain variable regions in order to retain the binding affinity and specificity of muDS6. Also included are versions exchanged for muDS6 surface residues defined by the corresponding asterisk residues in Table 1 (indicated by Kabat number) at one or more positions.

  [94] Disclosed herein are the primary amino acid and DNA sequences of the DS6 antibody light and heavy chains and humanized versions thereof. However, the scope of the present invention is not limited to antibodies and fragments comprising these sequences, but all antibodies and fragments that specifically bind to CA6 as a tumor-specific unique glycotope on the Mucl receptor. Included in the invention. Preferably, antibodies and fragments that specifically bind to CA6 antagonize the biological activity of the receptor. More preferably, such antibodies further have substantially no agonist activity. Thus, the antibodies and antibody fragments of the present invention are still within the scope of the present invention even though they differ in their backbone, CDR, and / or L and H chain amino acid sequences from the DS6 antibody or humanized derivative thereof. there is a possibility.

  [95] The CDRs of the DS6 antibody were identified by modeling and their molecular structure was deduced. Again, although CDRs are important for epitope recognition, they are not essential for the antibodies and antibody fragments of the invention. Thus, antibodies and fragments are provided that have improved properties, for example caused by affinity mutations of the antibodies of the invention.

  [96] Mouse L chain IgV from which DS6 appears to be derived? FIG. 11 shows the alignment of the ap4 germline gene and the heavy chain IgVh J558.41 germline gene with the DS6 antibody sequence. This comparison identifies possible somatic mutations in the DS6 antibody, including some in the CDRs.

  [97] The DS6 antibody heavy and light chain variable region sequences and the DS6 antibody CDR sequences have not been known so far and are shown in FIGS. 9A and 9B. Such information can be used to produce a humanized version of the DS6 antibody.

Antibody fragment
[98] Antibodies of the present invention include both full-length antibodies and epitope-binding fragments described above. As used herein, “antibody fragment” includes any portion of an antibody that retains the ability to bind to an epitope recognized by a full-length antibody, and is generally referred to as an “epitope binding fragment”. Examples of antibody fragments include Fab, Fab ′ and F (ab ′) ₂ , Fd, single chain Fv (scFv), single chain antibody, disulfide bond Fv (dsFv), and _VL or V _H regions. Fragments include but are not limited to these. Epitope binding fragments, including single chain antibodies, can include variable regions (one or more) alone or in combination with all or part of the following: hinge region, C _H 1, C _H 2, and C _H 3 domain.

[99] Such fragments can include one or both of Fab fragments or F (ab ′) ₂ fragments. Preferably, antibody fragments comprise all 6 CDRs of the entire antibody, although fragments comprising fewer than all such regions, eg, 3, 4 or 5 CDRs are also functional. In addition, these fragments may be members of any of the following immunoglobulin classes, or may bind those members: IgG, IgM, IgA, IgD, or IgE, and subclasses thereof.

[100] Fab and F (ab ′) ₂ fragments can be prepared by proteolytic cleavage using enzymes such as papain (Fab fragment) or pepsin (F (ab ′) ₂ fragment).

[101] The single chain FV (scFv) fragment comprises an epitope binding fragment comprising at least one fragment of an antibody heavy chain variable region (V _H ) linked to at least one fragment of an antibody light chain variable region (V _L ) It is. The linker ensures their proper three-dimensional folding when the (V _L ) and (V _H ) regions are joined, so as to maintain the target molecule binding specificity of the whole antibody from which the single chain antibody fragment is derived. It may be a short flexible peptide selected to occur. Carboxyl terminus of _{(V L)} or _{(V H)} sequences, the amino acid terminus of a complementary _{(V L)} or _{(V H)} sequence may be covalently linked by a linker.

[102] Single chain antibody fragments of the invention have at least one variable region or complementarity determining region (CDR) of all antibodies described herein, but some or all of the constant domains of these antibodies. Including the amino acid sequence. These constant domains are not required for antigen binding but constitute a major part of the structure of the whole antibody. Thus, single chain antibody fragments can overcome some of the problems associated with using antibodies that include some or all of the constant domains. For example, single chain antibody fragments tend to have no adverse interactions between biomolecules and heavy chain constant regions or other undesired biological activities. In addition, single chain antibody fragments are much smaller than whole antibodies and thus have greater capillary permeability than whole antibodies, so that single chain antibody fragments can more efficiently localize and bind to target antigen binding sites. Antibody fragments can also be produced on a relatively large scale in prokaryotic cells, thus facilitating their manufacture. Furthermore, relatively small sized single chain antibody fragments are less likely to elicit an immune response in the recipient than the whole antibody.

[103] Single chain antibody fragments can be prepared by molecular cloning, antibody phage display libraries, or similar methods well known to those skilled in the art. These proteins can be produced, for example, in eukaryotic cells or prokaryotic cells including bacteria. The epitope-binding fragments of the present invention can also be prepared by various phage display methods known in the art. In the phage display method, a functional antibody domain is displayed on the surface of a phage particle having a polynucleotide sequence encoding a functional antibody domain. In particular, such phage can be used to display epitope binding domains expressed from repertoire or combinatorial antibody libraries (eg, human or murine). Phage expressing an epitope binding domain that binds the antigen of interest can be selected or identified by antigen, for example by labeled antigen bound or captured on a solid surface or bead. Phages used in these methods generally include fd and M13 binding domains expressed from phage with Fab, Fv or disulfide stabilized Fv antibody domains recombinantly fused to either phage gene III or gene VIII protein. Filamentous phage.

[104] Examples of phage display methods that can be used to produce the epitope-binding fragments of the present invention include those disclosed below;

Each of these is incorporated herein in its entirety.
[105] After selecting the phage, the phage region encoding the fragment is isolated and selected in the selected host (including mammalian cells, insect cells, plant cells, yeast, and bacteria), for example, the set detailed below. An epitope-binding fragment can be produced by expressing it by a recombinant DNA method. For example, techniques for recombinant production of Fab, Fab ′ and F (ab ′) ₂ fragments by methods known in the art disclosed below can also be used; International Patent Application Publication WO 92/22324; Mullinax et al. , 1992, BioTechniques 12 (6): 864-869; Sawai et al., 1995, AJRI 34: 26-34; and Better et al., 1988, Science 240: 1041-1043; This is incorporated into the description. Examples of techniques that can be used to produce single chain Fv and antibodies include USP 4,946,778 and 5,258,498; Huston et al., 1991, Methods in Enzymology 203: 46-88; Shu et al., 1993, PNAS 90: 7995-7999; Skera et al., 1988, Science 240: 1038-1040.

Functional equivalent
[106] The present invention also includes functional equivalents of anti-CA6 antibodies and humanized anti-CA6 antibodies. The term “functional equivalent” includes, for example, antibodies with homologous sequences, chimeric antibodies, artificial antibodies and modified antibodies, where each functional equivalent is defined by its ability to bind to CA6. It will be appreciated by those skilled in the art that there is an overlap between a group of molecules called “antibody fragments” and a group called “functional equivalents”. Methods for producing functional equivalents are disclosed, for example, in International Patent Application Publication WO 93/21319; European Patent Application 239,400; International Patent Application Publication WO 89/09622; European Patent Application 338,745; and European Patent Application EP 332,424, Each of these is incorporated herein in its entirety.

[107] The antibody having a homologous sequence is an antibody having an amino acid sequence having sequence homology with the amino acid sequences of the anti-CA6 antibody of the present invention and the humanized anti-CA6 antibody. Preferably, the homology is homology with the amino acid sequences of the variable regions of the anti-CA6 antibodies of the present invention and humanized anti-CA6 antibodies. The “sequence homology” applied to the amino acid sequences in the present specification is determined by the FASTA search method according to, for example, Pearson and Lipman, Proc. Natl. Acad. Sci. USA, 85, 2444-2448 (1988). At least about 90%, 91%, 92%, 93
% Or 94% sequence homology, more preferably at least about 95%, 96%, 97%, 98%, or 99% sequence homology.

[108] A chimeric antibody as used herein is one in which different portions of the antibody are derived from different animal species. For example, an antibody having a variable region derived from a murine monoclonal antibody is paired with a human immunoglobulin constant region. Methods for producing chimeric antibodies are known in the art. See eg Morrison, 1985, Science 229: 1202; Oi et al., 1986, BioTechniques 4: 214; Gillies et al., 1989, J. Immunol. Methods 125: 191-202; USP 5,807,715; 4,816,567; and 4,816,397; All of which are incorporated herein by reference.

[109] Humanized forms of chimeric antibodies can be made, for example, by substituting the complementarity-determining regions of mouse antibodies into human framework domains; see, eg, International Patent Application Publication WO 92/22653. A humanized chimeric antibody is substantially or entirely derived from the constant region derived from the corresponding human antibody region and a variable region other than the complementarity determining region (CDR) and substantially or entirely from a non-human mammal. It has a CDR to do.

[110] Artificial antibodies include scFv fragments, diabodies, triabodies, tetrabodies and mru (Winter, G. and Milstein, C., 1991, Nature 349:
293-299; see review by Hudson, PJ, 1999, Current Opinion in Immunology 11: 548-557); each of which has antigen-binding ability. In the case of a single chain Fv fragment (scFv), the V _H and V _L domains of the antibody are linked by a flexible peptide. Generally, this linker peptide is about 15 amino acid residues long. If the linker is much smaller, for example 5 amino acids, a diabody is formed, which is a divalent scFv dimer. Shortening the linker to less than 3 amino acid residues results in the formation of trimeric and tetrameric structures called triabodies and tetrabodies. The minimum binding unit of an antibody is the CDR, generally the CDR2 of the heavy chain, which has sufficient specific recognition and binding capacity to be used separately. Such a fragment is called a molecular recognition unit, or mru. Several such mru can be joined with a short linker peptide, thus forming an artificial binding protein with a higher avidity than one mru.

[111] Functional equivalents of the present invention also include modified antibodies, eg, antibodies modified by covalently attaching any type of molecule to the antibody. For example, modified antibodies may be modified, for example, by glycosylation, pegylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, binding to cellular ligands or other proteins, etc. Antibodies are included. Covalent binding does not prevent the antibody from eliciting an anti-idiotypic response. These modifications can be performed by known methods, including but not limited to specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, and the like. Furthermore, the modified antibody can comprise one or more non-classical amino acids.

[112] Functional equivalents can be produced by exchanging different CDRs on different chains within different frameworks. For example, different heavy chain substitutions result in different classes of antibodies for a particular set of CDRs, which can produce, for example, IgG1-4, IgM, IgA1-2, IgD, IgE antibody types and isotypes. Similarly, artificial antibodies falling within the scope of the present invention can be produced by embedding a specific set of CDRs within a fully synthetic framework.

[113] A functional equivalent is a specific set of CDRs using a variety of methods known in the art.
Can be easily produced by mutation, deletion and / or insertion in the sequence of the variable region and / or constant region surrounding.

[114] Antibody fragments and functional equivalents of the present invention include molecules that have a degree of binding to CA6 that is detectable compared to the DS6 antibody. For detectable binding, a range of at least 10-100% of the binding capacity of the murine DS6 antibody to CA6, preferably at least 50%, 60% or 70%, more preferably at least 75%, 80%, 85% , 90%, 95% or 99% are all included.

Improved antibody
[115] CDRs are of primary importance for epitope recognition and antibody binding. However, the residues making up the CDR can be exchanged without interfering with the ability of the antibody to recognize and bind to its cognate epitope. For example, exchanges can be performed that increase the binding affinity of an antibody for an epitope without affecting epitope recognition.

[116] Accordingly, the scope of the invention includes improved versions of both murine and human antibodies that also specifically recognize CA6 and preferably bind with higher affinity.
[117] Some studies have shown that the introduction of one or more amino acid exchanges at various positions within an antibody sequence, based on knowledge of the primary antibody sequence, on the properties of the antibody, such as binding and expression levels. Searched (Yang, WP et al., 1995, J. Mol. Biol., 254, 392-403; Rader, C. et al., 1998, Proc. Natl. Acad. Sci. USA, 95: 8910-8915 Vaughan, TJ et al., 1998, Nature Biotechnology, 16, 535-539).

[118] In these studies, oligonucleotide-mediated site-directed mutagenesis,
Change the sequence of CDR1, CDR2, CDR3 or framework regions of heavy and light chain genes by methods such as cassette mutagenesis, error-prone PCR, DNA shuffling, or E. coli mutator strains This formed the equivalent of the primary antibody (Vaughan, TJ et al., 1998, Nature Biotechnology, 16, 535-539; Adey, NB et al., “Phage Display of Peptides and Proteins”, 1996, 16 Chapter, pp.277-291, editor Kay, BK et al., Academic Press). These methods of changing the sequence of the primary antibody improved the affinity of the secondary antibody.

[119] Anti-CA6 with improved functions, such as improved affinity for CA6, using the antibody sequences described herein in a similar specific manner that alters one or more amino acid residues of an antibody. Antibodies can be developed.

[120] Improved antibodies also include antibodies with improved properties produced by standard methods for immunization of animals, hybridoma formation, and selection of antibodies with specific properties.

Cytotoxic substances
[121] The cytotoxic agent used in the cytotoxic conjugates of the present invention may be any compound that leads to cell death, induces cell death, or decreases cell viability in some manner. Preferred cytotoxic agents include, for example, maytansinoids and maytansinoid analogs, taxoids, CC-1065 and CC-1065 analogs, dolastatin and dolastatin analogs as defined below. These cytotoxic agents are conjugated to the antibodies, antibody fragments, functional equivalents, improved antibodies and their analogs disclosed herein.

[122] Cytotoxic conjugates can be produced by in vitro methods. A linking group is used to attach a drug or prodrug to the antibody. Suitable linking groups are well known in the art and include disulfide groups, thioether groups, acid labile groups, photolabile groups, peptidase labile groups and esterase labile groups. Preferred linking groups are disulfide groups and thioether groups. For example, conjugates can be constructed using a disulfide exchange reaction or by forming a thioether bond between an antibody and a drug or prodrug.

Maytansinoids
[123] Cytotoxic agents that can be used in the present invention to form cytotoxic conjugates include maytansinoids and maytansinoid analogs. Examples of suitable maytansinoids include maytansinol and maytansinol analogues. Maytansinoids are drugs that inhibit microtubule formation and are highly toxic to mammalian cells.

[124] Examples of suitable maytansinols include those with modified aromatic rings and those with modifications at other positions. Such suitable maytansinoids include the following USP
It is described in:

[125] Specific examples of suitable maytansinol analogs with modified aromatic rings include the following:
[126] (1) C-19-dechloro (USP 4,256,746) (produced by LAH reduction of ansamytocin P2);
[127] (2) C-20-hydroxy (or C-20-demethyl) +/- C-19-
Dechloro (USP 4,361,650 and 4,307,016) (produced by demethylation using Streptomyces or Actinomyces, or dechlorination using LAH); and
[128] (3) C-20-demethoxy, C-20-acyloxy (-OCOR) +/-
Dechloro (USP 4,294,757) (prepared by acylation with acyl chloride).

[129] Specific examples of suitable maytansinol analogs with modifications at other positions include the following:
[130] (1) (prepared by reaction of maytansinol with _{H 2} S or _{P 2 S 5) C-9} -SH (USP 4,424,219);
[131] (2) C-14-alkoxymethyl (demethoxy / CH ₂ OR) (USP 4,331,5
98);
[132] (3) C- 14- hydroxymethyl or acyloxymethyl _(CH 2 OH or _{CH 2 OAc) (USP 4,450,254)} ( prepared from Nocardia sp (Nocardia));
[133] (4) C-15-hydroxy / acyloxy (USP 4,364,866) (produced by conversion of maytansinol by Streptomyces);
[134] (5) C-15-methoxy (USP 4,313,946 and 4,315,929) (isolated from Trewia nudiflora);
[135] (6) C-18-N-demethyl (USP 4,362,663 and 4,322,348) (produced by demethylation of maytansinol by Streptomyces); and
[136] (7) 4,5-Deoxy (USP 4,371,533) (produced by reduction of maytansinol in titanium trichloride / LAH).

In [137] preferred embodiment, the cytotoxic conjugates of the present invention, the thiol-containing maytansinoid (DM1), formally N ^{2 '-} deacetyl -N ^2' - (3- mercapto-1-oxopropyl) - maytansine Is used as a cytotoxic substance. DM1 is represented by the following structural formula (I):

In [138] Another preferred embodiment, the cytotoxic conjugates of the present invention, the thiol-containing maytansinoid N ^{2 '-} deacetyl -N ^2' - (4-methyl-4-mercapto-1-oxopentyl) - maytansine Used as a cytotoxic substance. DM4 is represented by the following structural formula (II):

[139] Other maytansines can be used in other embodiments of the present invention, including thiol- and disulfide-containing maytansinoids with mono- or di-alkyl substitution at the carbon atom bearing the sulfur atom. These include maytansinoids having the following: C-3, C-14 hydroxymethyl, C-15 hydroxy, or C-20 desmethyl, acylated amino acid side chains in which the acyl group carries a hindered sulfhydryl group Wherein the carbon atom of the acyl group carrying the thiol functional group has 1 or 2 substituents, the substituents being CH 3, C 2 H 5, linear or branched alkyl having 1 to 10 carbon atoms or An alkenyl, a cyclic alkyl or alkenyl having 3 to 10 carbon atoms, phenyl, substituted phenyl, or a heterocyclic aromatic or heterocycloalkyl group, and one of the substituents may be H; Acyl groups have a straight chain of at least 3 carbon atoms in length between the carbonyl function and the sulfur atom.

[140] Such other maytansines include compounds represented by formula (III):

In the formula:
Y '

Wherein R ₁ and R ₂ are each independently CH ₃ , C ₂ H ₅ , a linear alkyl or alkenyl having 1 to 10 carbon atoms, and a moiety having 3 to 10 carbon atoms. A branched or cyclic alkyl or alkenyl, phenyl, substituted phenyl, or a heterocyclic aromatic or heterocyclic group, and R ₂ may be H;
A, B, D are cycloalkyl or cycloalkenyl having 3 to 10 carbon atoms, simple or substituted aryl or heterocyclic aromatic or heterocyclic group;
R ₃ , R ₄ , R ₅ , R ₆ , R ₇ , R ₈ , R ₉ , R ₁₀ , R ₁₁ and R ₁₂ are each independently H, CH ₃ , C ₂ H ₅ , 1 to 10 A linear alkyl or alkenyl having 3 carbon atoms, a branched or cyclic alkyl or alkenyl having 3 to 10 carbon atoms, phenyl, substituted phenyl, or a heterocyclic aromatic or heterocyclic group;
l, m, n, o, p, q, r, s, t and u are each independently an integer of 0 or 1 to 5, provided that l, m, n, o, p, q, r, at least two of s, t and u are not zero in any case;
Z is H, SR or —COR, wherein R is a straight chain alkyl or alkenyl having 1 to 10 carbon atoms, a branched or cyclic alkyl or alkenyl having 3 to 10 carbon atoms, Or a simple or substituted aryl or heterocyclic aromatic or heterocyclic group.

[141] Preferred embodiments of formula (III) include compounds of formula (III):
R ₁ is methyl, R ₂ is H, and Z is H;
R ₁ and R ₂ are methyl and Z is H;
R ₁ is methyl, R ₂ is H, and Z is —SCH ₃ ;
R ₁ and R ₂ are methyl and Z is —SCH ₃ .

[142] Such other maytansines include those of formula (IV-L), (IV-D) or (
Also included are compounds represented by IV-D, L):

In the formula:
Y is

Wherein R ₁ and R ₂ are each independently CH ₃ , C ₂ H ₅ , a linear alkyl or alkenyl having 1 to 10 carbon atoms, and a moiety having 3 to 10 carbon atoms. A branched or cyclic alkyl or alkenyl, phenyl, substituted phenyl, or a heterocyclic aromatic or heterocyclic group, and R ₂ may be H;
R ₃ , R ₄ , R ₅ , R ₆ , R ₇ and R ₈ are each independently H, CH ₃ , C ₂ H ₅ , linear alkyl or alkenyl having 1 to 10 carbon atoms, 3 A branched or cyclic alkyl or alkenyl having 10 to 10 carbon atoms, phenyl, substituted phenyl, or a heterocyclic aromatic or heterocyclic group;
l, m and n are each independently an integer of 1 to 5, and n may be 0;
Z is H, SR or —COR, wherein R is a linear or branched alkyl or alkenyl having 1 to 10 carbon atoms, a cyclic alkyl or alkenyl having 3 to 10 carbon atoms, Or a simple or substituted aryl or heterocyclic aromatic or heterocyclic group;
May represents a maytansinoid having a C-3 side chain, C-14 hydroxymethyl, C-15 hydroxy, or C-20 desmethyl.

[143] Preferred embodiments of formulas (IV-L), (IV-D) and (IV-D, L) include:
The following compounds of formula (IV-L), (IV-D) and (IV-D, L) are included:
R ₁ is methyl, R ₂ is H, R ₅ , R ₆ , R ₇ and R ₈ are each H, l and m are each 1, n is 0, Z is H is there;
R ₁ and R ₂ are methyl, R ₅ , R ₆ , R ₇ and R ₈ are each H, l and m are 1, n is 0, and Z is H;
R ₁ is methyl, R ₂ is H, R ₅ , R ₆ , R ₇ and R ₈ are each H, l and m are each 1, n is 0, and Z is —SCH ₃ ;
R ₁ and R ₂ are methyl, R ₅ , R ₆ , R ₇ and R ₈ are each H, l and m are 1, n is 0, and Z is —SCH ₃ .

[144] Preferably, the cytotoxic substance is represented by the formula (IV-L).
[145] Such other maytansines also include compounds represented by formula (V):

In the formula:
Y is

Wherein R ₁ and R ₂ are each independently CH ₃ , C ₂ H ₅ , a linear alkyl or alkenyl having 1 to 10 carbon atoms, and a moiety having 3 to 10 carbon atoms. A branched or cyclic alkyl or alkenyl, phenyl, substituted phenyl, or a heterocyclic aromatic or heterocyclic group, and R ₂ may be H;
R ₃ , R ₄ , R ₅ , R ₆ , R ₇ and R ₈ are each independently H, CH ₃ , C ₂ H ₅ , linear alkyl or alkenyl having 1 to 10 carbon atoms, 3 A branched or cyclic alkyl or alkenyl having 10 to 10 carbon atoms, phenyl, substituted phenyl, or a heterocyclic aromatic or heterocyclic group;
l, m and n are each independently an integer of 1 to 5, and n may be 0;
Z is H, SR or —COR, wherein R is a straight chain alkyl or alkenyl having 1 to 10 carbon atoms, a branched or cyclic alkyl or alkenyl having 3 to 10 carbon atoms, Or a simple or substituted aryl or heterocyclic aromatic or heterocyclic group.

[146] Preferred embodiments of formula (V) include compounds of formula (V):
R1 is methyl, R2 is H, R5, R6, R7 and R8 are each H; l and m are each 1; n is 0; Z is H;
R ₁ and R ₂ are methyl; R ₅ , R ₆ , R ₇ and R ₈ are each H; l and m are 1; n is 0; Z is H;
R ₁ is methyl, R ₂ is H, R ₅ , R ₆ , R ₇ and R ₈ are each H, l and m are each 1, n is 0, and Z is —SCH ₃ ;
R ₁ and R ₂ are methyl, R ₅ , R ₆ , R ₇ and R ₈ are each H, l and m are 1, n is 0, and Z is —SCH ₃ .

[147] Such other maytansines further include compounds of the formula (VI-L), (VI-D) or (VI-D, L):

In the formula:
Y ₂ is,

Wherein R ₁ and R ₂ are each independently CH ₃ , C ₂ H ₅ , a linear alkyl or alkenyl having 1 to 10 carbon atoms, and a moiety having 3 to 10 carbon atoms. A branched or cyclic alkyl or alkenyl, phenyl, substituted phenyl, or a heterocyclic aromatic or heterocyclic group, and R ₂ may be H;
R ₃ , R ₄ , R ₅ , R ₆ , R ₇ and R ₈ are each independently H, CH ₃ , C ₂ H ₅ , linear cyclic alkyl or alkenyl having 1 to 10 carbon atoms. A branched or cyclic alkyl or alkenyl having 3 to 10 carbon atoms, phenyl, substituted phenyl, or a heterocyclic aromatic or heterocyclic group;
l, m and n are each independently an integer of 1 to 5, and n may be 0;
Z ₂ is SR or COR, where R is a linear alkyl or alkenyl having 1 to 10 carbon atoms, a branched or cyclic alkyl or alkenyl having 3 to 10 carbon atoms, or simply Or a substituted aryl or heterocyclic aromatic or heterocyclic group;
May is a maytansinoid.

[148] Such other maytansines also include compounds represented by formula (VII):

In the formula:
Y ₂ '

Wherein R ₁ and R ₂ are each independently CH ₃ , C ₂ H ₅ , straight chain branched alkyl or alkenyl having 1 to 10 carbon atoms, and 3 to 10 carbon atoms. A cyclic alkyl or alkenyl having a phenyl, a substituted phenyl, or a heterocyclic aromatic or heterocyclic group, and R ₂ may be H;
A, B and D are each independently a cycloalkyl or cycloalkenyl, simple or substituted aryl or heteroaromatic or heterocyclic group having 3 to 10 carbon atoms;
R ₃ , R ₄ , R ₅ , R ₆ , R ₇ , R ₈ , R ₉ , R ₁₀ , R ₁₁ and R ₁₂ are each independently H, CH ₃ , C ₂ H ₅ , 1 to 10 A linear alkyl or alkenyl having 3 carbon atoms, a branched or cyclic alkyl or alkenyl having 3 to 10 carbon atoms, phenyl, substituted phenyl, or a heterocyclic aromatic or heterocyclic group;
l, m, n, o, p, q, r, s, t and u are each independently an integer of 0 or 1 to 5, provided that l, m, n, o, p, q, r, at least two of s, t and u are not zero in any case;
Z ₂ is SR or —COR, wherein R is a linear alkyl or alkenyl having 1 to 10 carbon atoms, a branched or cyclic alkyl or alkenyl having 3 to 10 carbon atoms, or A simple or substituted aryl or heterocyclic aromatic or heterocyclic group.

[149] In a preferred embodiment of formula (VII), formula (VII) wherein R1 is methyl and R2 is H
VII).
[150] The maytansinoids described above can be conjugated to the anti-CA6 antibody DS6 or a homologue or fragment thereof. In that case, C-3 of maytansinoid
The thiol- or disulfide functional group, C-14 hydroxymethyl, C-15 hydroxy, or C-20 desmethyl in the acyl group of the acylated amino acid side chain in The thiol- or disulfide functional group of the acyl group of the acylated amino acid side chain is on a carbon atom with one or two substituents, which substituents are CH ₃ , C ₂ H ₅ , 1-10 carbon atoms. A straight-chain alkyl or alkenyl having a branched chain or cyclic alkyl or alkenyl having 3 to 10 carbon atoms, phenyl, substituted phenyl, or heterocyclic aromatic or heterocycloalkyl group, One may be H and the acyl group is at least 3 carbon atoms between the carbonyl function and the sulfur atom. With a linear chain of length.

[151] Preferred conjugates of the invention are those comprising an anti-CA6 antibody DS6 conjugated to a maytansinoid of formula (VIII) or a homologue or fragment thereof:

In the formula:
Y ₁ '

Wherein A, B and D are each independently a cycloalkyl or cycloalkenyl having 3 to 10 carbon atoms, a simple or substituted aryl or a heterocyclic aromatic or heterocyclic group. Yes;
R ₃ , R ₄ , R ₅ , R ₆ , R ₇ , R ₈ , R ₉ , R ₁₀ , R ₁₁ and R ₁₂ are each independently H, CH ₃ , C ₂ H ₅ , 1 to 10 A linear alkyl or alkenyl having 3 carbon atoms, a branched or cyclic alkyl or alkenyl having 3 to 10 carbon atoms, phenyl, substituted phenyl, or a heterocyclic aromatic or heterocyclic group;
l, m, n, o, p, q, r, s, t and u are each independently an integer of 0 or 1 to 5, provided that l, m, n, o, p, q, r, At least two of s, t and u are not non-zero in any case.

[152] Preferably, R ₁ is methyl and R ₂ is H, or R ₁ and R ₂
Is methyl.
[153] A more preferred conjugate of the invention is an anti-CA6 antibody DS6 conjugated to a maytansinoid of formula (IX-L), (IX-D) or (IX-D, L) or a homologue or fragment thereof Including:

In the formula:
Y ₁ is,

Wherein R ₁ and R ₂ are each independently CH ₃ , C ₂ H ₅ , a linear alkyl or alkenyl having 1 to 10 carbon atoms, and a moiety having 3 to 10 carbon atoms. A branched or cyclic alkyl or alkenyl, phenyl, substituted phenyl, or a heterocyclic aromatic or heterocyclic group, and R ₂ may be H;
R ₃ , R ₄ , R ₅ , R ₆ , R ₇ and R ₈ are each independently H, CH ₃ , C ₂ H ₅ , linear alkyl or alkenyl having 1 to 10 carbon atoms, 3 A branched or cyclic alkyl or alkenyl having 10 to 10 carbon atoms, phenyl, substituted phenyl, or a heterocyclic aromatic or heterocyclic group;
l, m and n are each independently an integer of 1 to 5, and n may be 0;
May represents a maytansinoid having a C-3 side chain, C-14 hydroxymethyl, C-15 hydroxy, or C-20 desmethyl.

[154] Preferred embodiments of formulas (IX-L), (IX-D) and (IX-D, L) include:
The following compounds of formula (IX-L), (IX-D) and (IX-D, L) are included:
R ₁ is methyl and R ₂ is H, or R ₁ and R ₂ are methyl;
R ₁ is methyl, R ₂ is H, R ₅ , R ₆ , R ₇ and R ₈ are each H; l and m are each 1; n is 0;
R ₁ and R ₂ are methyl; R ₅ , R ₆ , R ₇ and R ₈ are each H; l and m are 1; n is 0.

[155] Preferably, the cytotoxic substance is represented by formula (IX-L).
[156] More preferred conjugates of the invention are those comprising an anti-CA6 antibody DS6 conjugated to a maytansinoid of formula (X) or a homologue or fragment thereof:

Substituents in the formula are as defined for formula (IX) above.
[157] Particularly preferred are those in which R ₁ is H, R ₂ is methyl, R ₅ , R ₆ , R ₇ and R ₈ are each H, and l and m are each 1 , N is 0.

[158] Further particularly preferred are those in which R ₁ and R ₂ are methyl, R ₅ , R ₆ , R ₇ and R ₈ are each H, l and m are 1 and n is 0 It is a compound which is.

[159] Furthermore, L-aminoacyl stereoisomers are preferred.
[160] Each maytansinoid taught in pending US patent application 10 / 849,136 (filed May 20, 2004) can also be used in the cytotoxic conjugates of the present invention. The entire disclosure of US patent application 10 / 849,136 is incorporated herein by reference.

Linking group containing disulfide
[161] In order to link maytansinoids to cell binding agents, eg, DS6 antibodies, maytansinoids include a linking moiety. The linking moiety includes a chemical bond that can release fully effective maytansinoids at specific sites. Suitable chemical bonds are well known in the art and include disulfide bonds, acid labile bonds, photolabile bonds, peptidase labile groups and esterase labile groups. Preferred is a disulfide bond.

[162] The linking moiety also includes a reactive chemical group. In preferred embodiments, the reactive chemical group can be covalently attached to the maytansinoid via a disulfide bond linking moiety.
[163] Particularly preferred reactive chemical groups are N-succinimidyl esters and N-sulfosuccinimidyl esters.

[164] Particularly preferred maytansinoids comprising a linking group having a reactive chemical group are C-3 esters of maytansinol and its analogs, wherein the linking moiety comprises a disulfide bond, and the reactive chemical group is N-succinimidyl ester or N-sulfosuccinimidyl ester.

[165] Numerous positions of maytansinoids can be used as positions for chemical linking of linking moieties. For example, the C-3 position with a hydroxy group, the C-14 position modified with hydroxymethyl, the C-15 position modified with hydroxy, and the C-20 position with a hydroxy group are all expected to be useful. . However, the C-3 position is preferred, and the C-3 position of maytansinol is particularly preferred.

[166] Although the synthesis of maytansinol esters with linking moieties is described in connection with linking moieties that contain disulfide bonds, other chemical bonds (as described above) can be used, as can other maytansinoids. It will be appreciated by those skilled in the art that a connecting portion having a Examples of other chemical bonds include acid labile bonds, photolabile bonds, peptidase labile groups and esterase labile groups. USP 5,208,020 (incorporated herein)
Discloses the preparation of maytansinoids having such bonds.

[167] The synthesis of maytansinoids and maytansinoid derivatives having a disulfide moiety with a reactive group is described in USP 6,441,163 and 6,333,410, and US patent application 10 / 161,651, each incorporated herein by reference. .

[168] Maytansinoids containing reactive groups, such as DM1, can be combined with antibodies, such as DS
React with 6 antibody to produce a cytotoxic conjugate. These conjugates can be purified by HPLC or gel filtration.

[169] Several excellent methods for producing such antibody-maytansinoid conjugates are presented in USP 6,333,410, and US patent applications 09 / 867,598, 10 / 161,651 and 10 / 024,290, Each of which is incorporated herein in its entirety.

[170] In general, antibody solutions in aqueous buffer may be incubated with an excess of maytansinoids having a disulfide moiety with a reactive group. The reaction mixture can be quenched by the addition of excess amine (eg ethanolamine, taurine, etc.). The maytansinoid-antibody conjugate can then be purified by gel filtration.

[171] The number of maytansinoids bound per antibody molecule is 252 nm and 28
Determination can be made by measuring the ratio of absorbance at 0 nm by spectrophotometry. An average of 1-10 maytansinoid molecules / antibody molecules is preferred.

[172] Conjugates of antibodies and maytansinoid drugs can be evaluated in vitro for their ability to inhibit the growth of a variety of adverse cell lines. For example, cell lines such as the human epidermoid carcinoma cell line A-431, the human small cell lung cancer cell line SW2, the human breast tumor cell line SKBR3, and the Burkitt lymphoma cell line Namalwa can be used to facilitate the cytotoxicity of these compounds. Can be evaluated. The cells to be evaluated can be exposed to the compounds of the present invention for 24 hours, and the viability of the cells can be measured by a direct assay by a known method. IC ₅₀ values can then be calculated from the assay results.

Linking group containing PEG
[173] Maytansinoids can also be attached to cell binding agents using PEG linking groups; described in US patent application Ser. No. 10 / 024,290. These PEG linking groups are soluble in both water and non-aqueous solvents and can be used to attach one or more cytotoxic agents to the cell binding agent. Examples of PEG linking groups include heterobifunctional PEG linkers, which are at the opposite end of the linker, at one end by a functional sulfhydryl group or disulfide group, at the other end by an active ester, and a cytotoxic agent. Bind to cell binding agent.

[174] As a general example of the synthesis of cytotoxic conjugates using PEG linking groups, US patent application 10 / 024,290 is again incorporated by reference for specific details. Synthesis is initiated by reacting one or more cytotoxic substances having a reactive PEG moiety with a cell binding agent, replacing the terminal active ester of each reactive PEG moiety with an amino acid residue of the cell binding agent, and PEG A cytotoxic conjugate is obtained comprising one or more cytotoxic agents covalently bound to the cell binding agent by a linking group.

Taxanes
[175] The cytotoxic agent used in the cytotoxic conjugate according to the present invention may be a taxane or a derivative thereof.

[176] Taxanes consist of two compounds, the natural cytotoxic product paclitaxel (Taxol) and the semisynthetic derivative docetaxel (Taxotere), widely used in the treatment of cancer. It is a compound group including. Taxanes are mitotic spindle toxins that inhibit tubulin open polymerization and lead to cell death. Although docetaxel and paclitaxel are useful drugs for the treatment of cancer, their anti-tumor activity is limited due to their nonspecific toxicity to normal cells. In addition, compounds such as paclitaxel and docetaxel themselves are not effective enough for use in cell binding agent conjugates.

[177] Preferred taxanes for use in the manufacture of cytotoxic conjugates are of the formula (XI
) Is a taxane:

[178] Methods for synthesizing taxanes that can be used in the cytotoxic conjugates of the invention, and for conjugating taxanes to cell binding agents, such as antibodies, are described in USP 5,416,064, 5,475,092, 6,340,701, 6,372,738 and 6,436,931, and the United States. Details are described in patent applications 10 / 024,290, 10 / 144,042, 10 / 207,814, 10 / 210,112 and 10 / 369,563.

CC-1065 analog
[179] The cytotoxic agent used in the cytotoxic conjugate according to the present invention is CC-
It may be 1065 or a derivative thereof.

[180] CC-1065 is an effective antitumor antibiotic isolated from a culture broth of Streptomyces zelensis. CC-1065 is about 1000 times more effective in vitro than conventional anticancer drugs such as doxorubicin, methotrexate and vincristine (BK Bhuyan et al., Cancer Res., 42, 3532-3537
(1982)). CC-1065 and its analogs are USP 6,372,738, 6,340,701, 5,846,545.
And 5,585,499.

[181] The cytotoxic potency of CC-1065 correlates with its alkylating activity and its DNA binding or DNA intercalation activity. These two activities are in separate parts of the molecule. For example, the alkylating activity is contained in a cyclopropyroloindole (CPI) subunit and the DNA binding activity is in two pyrroloindole subunits.

[182] CC-1065 has certain attractive features as a cytotoxic agent, but has limited therapeutic use. Administration of CC-1065 to mice resulted in delayed hepatotoxicity and died on day 50 after a single intravenous dose of 12.5 μg / kg {VL Reynolds et al., J. Antibiotics, XXIX, 319- 334 (1986)}. This spurred an attempt to develop an analog that did not cause delayed toxicity, and a simpler analog modeled on CC-1065 was reported {MA Warpehoski et al., J. Med. Chem., 31 , 590-603 (1988)}.

[183] In other series of analogs, the CPI moiety is cyclopropabenzoindole (CBI).
) Moiety substituted {DLBoger et al., J. Org. Chem., 55, 5823-5833 (1990); DL Boger et al., BioOrg. Med. Chem. Lett., 1, 115-120 (1991 )}. These compounds are
It retains the high in vitro titer of the parent compound without causing delayed toxicity in mice. Similar to CC-1065, these compounds are alkylating agents that bind to the minor groove of DNA in a covalent manner, leading to cell death. However, clinical evaluation of the most promising analogs, Adozelesin and Carzelesin, has had disappointing results {BFFoster et al., Investigational New Drugs, 13, 321-326 (1996); I. Wolff et al., Clin. Cancer Res., 2, 1717-1723 (1996)}. These drugs show a low therapeutic effect due to their high systemic toxicity.

[184] The therapeutic efficacy of CC-1065 analogs is achieved by altering the in vivo distribution by targeting and delivering to the tumor site, thereby reducing toxicity to off-target tissues and thereby reducing systemic toxicity. Can be greatly improved. To achieve this goal, conjugates of CC-1065 analogs and derivatives and cell binding agents that specifically target tumor cells have been reported {USP; 5,475,092; 5,585,499; 5,846,545}. These conjugates generally show high target-specific toxicity in vitro and marked antitumor activity in mouse human tumor xenograft models {RVJ Chari et al., Cancer Res., 55, 4079-4084 (1995) }.

[185] Methods for synthesizing CC-1065 analogs that can be used in the cytotoxic conjugates of the present invention, and for conjugating these analogs to cell binding agents, such as antibodies, are described in USP 5,475,092, 5,846,545, 5,585,499, 6,534,660 and 6,586,618 and US patent applications 10 / 116,053 and 10 / 265,452.

Other drugs
[186] methotrexate, daunorubicin, doxorubicin, vincristine, vinblastine, melphalan, mitomycin C, and chlorambucil, calicheamicin, tubulysin and tubricin analogues, duocarmycin and duocarmycin analogues , Dolastatin and dolastatin analogs are also suitable for preparing the conjugates of the invention. These drug molecules can also be linked to the antibody molecule by an intermediate carrier molecule, such as serum albumin. For example, doxalubicin and daunorubicin compounds described in US patent application 09/740991 would also be useful cytotoxic agents.

Therapeutic composition
[187] The present invention also provides a therapeutic composition comprising:
(A) an effective amount of one or more cytotoxic conjugates, and (b) a pharmaceutically acceptable carrier.

[188] The present invention provides a method for inhibiting the growth of a specific cell population comprising a target cell or a tissue comprising a target cell and an effective amount of a cytotoxic conjugate, or a cytotoxic conjugate alone or Also provided is a method comprising contacting a therapeutic agent comprising in combination with another cytotoxic agent or therapeutic agent.

[189] The present invention also includes a method of treating a subject with cancer using the therapeutic composition of the present invention.
[190] Cytotoxic conjugates can be evaluated for in vitro potency and specificity by methods previously described (eg RVJ Chari et al., Cancer Res., 55, 4079-4084 (1995) See). Anti-tumor activity can be similarly assessed in mouse human tumor xenograft models by the methods previously described (eg, Liu et al., Proc. Natl. Acad. Sci. 93: 8618-8623 ( 1996)).

[191] Suitable pharmaceutically acceptable carriers are well known and can be determined by one skilled in the art based on the clinical situation. Carriers as used herein include diluents and excipients.
[192] Examples of suitable carriers, diluents and / or excipients include: (1) Dulbecco's phosphate buffered saline, pH about 7.4, 1-25 mg / ml. With or without human serum albumin, (2) 0.9% saline (0.9% w / v sodium chloride (NaCl)), and (3) 5% (w / v) Of dextrose; an antioxidant such as tryptamine and the stabilizer Tween 20.

[193] The method of inhibiting the growth of a particular cell population can be performed in vitro, in vivo, or ex vivo. Growth inhibition, as used herein, delays cell growth, reduces cell viability, leads to cell death, lyses cells, and cell death, whether short or long term Means to trigger.

[194] Examples of in vitro uses include: treatment to kill diseased or malignant cells before transplanting autologous bone marrow into the same patient; killing and transplanting competent T cells before transplanting bone marrow Treatment to prevent unpaired host disease (GVHD); treatment of cell cultures to kill all cells that do not express the target antigen other than the target variant, or to kill variants that express unwanted antigens .

[195] Non-clinical in vitro use conditions can be readily determined by one skilled in the art.
[196] Examples of clinical ex vivo use are removal of tumor cells or lymphoid cells from the bone marrow prior to autotransplantation in the treatment of cancer or autoimmune disease, or prevent graft-versus-host disease (GVHD) Therefore, the removal of competent T cells and other lymphoid cells from autologous or allogeneic bone marrow or tissue prior to transplantation. The treatment can be carried out according to the following. Bone marrow is harvested from a patient or other individual and then incubated in serum-containing medium supplemented with the cytotoxic agents of the present invention. Concentration is about 10 μM to 1 pM, about 30 minutes to about 48 hours, 37 ° C. The exact conditions of concentration and incubation time, i.e. dose, can be easily determined by one skilled in the art. After incubation, bone marrow cells are washed with serum-containing medium and returned to the patient by intravenous infusion according to known methods. If the patient undergoes other treatments, such as ablation chemotherapy or whole body irradiation, between bone marrow collection and treatment cell reinfusion, the treated bone marrow cells are placed in liquid nitrogen using standard medical equipment. Keep frozen.

[197] For clinical in vivo use, a cytotoxic conjugate of the invention is prepared as a solution, which is tested for sterility and endotoxin concentration. Examples of suitable protocols for administration of cytotoxic conjugates are as follows: The conjugate is administered as an intravenous bolus once a week for 4 weeks. A bolus dose may be administered in 50-100 ml saline, to which 5-10 ml human serum albumin may be added. The dose is 10 μg to 100 mg per intravenous dose (100 ng to 1 mg / kg per day). More preferably, the dosage is 50 μg to 30 mg. Most preferably, the dosage is 1-20 mg. After 4 weeks of treatment, patients can continue to receive baseline treatment once a week. Specific clinical protocols regarding routes of administration, excipients, diluents, dosages, times, etc. can be determined by one skilled in the art based on the clinical situation.

[198] Examples of pathological conditions that can be treated by in vivo or ex vivo methods to kill specific cell populations include any type of malignancy including, for example, lung cancer, breast cancer, colon cancer Prostate cancer, renal cancer, pancreatic cancer, ovarian cancer, cervical cancer and lymphoid cancer, osteosarcoma, synovial cancer, sarcoma or cancer expressing CA6, and other cancers mainly expressing CA6 glycotope Autoimmune diseases such as systemic lupus, rheumatoid arthritis, and multiple sclerosis; transplant rejection, such as renal transplant rejection, liver transplant rejection, lung transplant rejection, heart transplant rejection And bone marrow transplant rejection; graft-versus-host disease; viral infections such as mV infection, HIV infection, AIDS, etc .; and parasitic infections such as giardiasis, amebiasis, Blood schistosomiasis, as well as other diseases that those skilled in the art to determine.

kit
[199] The invention also includes kits comprising, for example, the cytotoxic conjugates described above and instructions for using the cytotoxic conjugates to kill a particular cell type. The instructions include instructions for using the cytotoxic conjugate in vitro, in vivo or ex vivo.

[200] In general, kits are equipped with compartments for containing cytotoxic conjugates. The cytotoxic conjugate may be in lyophilized form, liquid form or other form that can be contained in the kit. The kit is combined with other components necessary to carry out the methods described in the instructions in the kit, such as a sterile solution for reconstitution of the lyophilized powder, with a cytotoxic conjugate prior to administration to the patient Other agents and devices that assist in administering the conjugate to the patient may be housed.

Other aspects
[201] The present invention further provides monoclonal antibodies, humanized antibodies, and epitope-binding fragments thereof that are further labeled for use in research or diagnostic applications. In preferred embodiments, the label is a radioactive label, fluorophore, chromophore, contrast agent or metal ion.

[202] A diagnostic method is also provided in which a labeled antibody or epitope-binding fragment thereof is administered to a subject suspected of having cancer and the distribution of the label in the subject's body is measured or monitored.

Example
[203] The broad scope of this invention is best understood with reference to the following examples. These are not intended to limit the invention to any particular embodiment.

Example 1: Identification of antigen positive and negative cell lines by flow cytometry binding assay
[204] CA6, a DS6 epitope, was localized to the cell surface using flow cytometry analysis. Human cell lines were obtained from the American Type Culture Collection (ATCC) except for the following: OVCAR5 cells (Kearse et al., Int. J. Cancer 88 (6): 866-872 (2000)), OVCAR8 And IGROV1 cells (M. Seiden, Massachusetts General Hospital). All cells were treated with 4 mM L-glutamine, 50 U / ml penicillin, 50 μg / ml streptomycin (Cambrex Bio Science, Rockland, Maine) and 10% v / v fetal calf serum (Atlas Biologicals, Fort Fort, Colorado). The cells were grown in RPMI 1640 (hereinafter referred to as medium) supplemented with Collins). Cells were maintained at 37 ° C., 5% CO _{2 in} a humidified incubator.

[205] Cells (1-2 × 10 ⁻⁵ cells / well) were prepared in serial dilutions of DS6 prepared in FACS buffer (2% goat serum, RPMI) in 96-well plates for 3-4 hours on ice. Incubated with antibody. The cells were centrifuged for 5 minutes at 1500 rpm and 4 ° C. using a tabletop centrifuge. After removing the medium, the wells were then refilled with 150 μl FACS buffer. This washing step was then repeated. FITC-labeled goat anti-mouse IgG (Jackson Immunoresearch) was diluted 1: 100 in FACS buffer and incubated with cells for 1 hour on ice. The plate was covered with foil to prevent signal loss. After two washes, the cells were fixed with 1% formaldehyde and analyzed with a flow cytometer.

[206] As expected from tumor immunohistochemistry, cell lines derived from the ovary, breast, cervix and pancreas mainly showed CA6 epitopes (Table 3). However, CA6 expression exhibited by some cell lines of other tumor types has been limited. DS6 antibody binds with an apparent _{K D} of 135.6PM (in PC-3 cells, Table 3). The maximum average fluorescence (Table 3) of the binding curve (FIG. 1) in antigen positive cells suggests the relative density of the antigen.

^* Average maximum relative average fluorescence
Example 2: Characterization of the DS6 epitope
[207] Antigens of DS6 by immunoblotting a dot blot of CA6 positive cell lysate (Caov-3) digested by proteolytic (pronase and proteinase K) and / or glycolytic (neuraminidase and periodic acid) treatments The characteristics of CA6 were analyzed. As a positive control, other antibodies recognizing various types of epitopes were tested for lysates of antigen positive cell lines (Cavo-3 and CM1; Colo205 and C242; SKMEL28 and R24). CM1 is an antibody that recognizes the protein epitope of the variable number tandem repeat domain (VNTR) of Muc1, and thus serves as a control for the protein epitope. C242 binds to a novel colorectal cancer-specific sialic acid-dependent glycotope (CanAg) on Mucl and serves as a control for glycotope on protein. R24 binds to the GD3 ganglioside specific for melanoma and is therefore a control for glycotopes on the non-protein backbone.

[208] Caov-3, Colo205 and SKMEL28 cells were seeded into 15 cm tissue culture plates. The medium (30 mL / plate) was changed the day before cell lysis. Modified RIPA buffer (50 mM Tris-HCl, pH 7.6, 150 mM NaCl, 5 mM EDTA, 1% NP40, 0.25% sodium deoxycholate), protease inhibitors (PMSF, pepstatin A, leupeptin, And aprotinin), and PBS were pre-cooled on ice. After the medium was aspirated from the plate, the cells were washed twice with 10 ml cold PBS. All subsequent steps were performed on ice and / or in a cold room at 4 ° C. After the final PBS wash, the cells were washed with 1-2 mL of lysis buffer (protease inhibitor added to RIPA buffer, final concentration 1 mM PMSF, 1 μM pepstatin A, 10 μm
g / ml leupeptin and 2 μg / ml aprotinin). The lysate was scraped from the plate with a cell lifter and triturated by aspirating and discharging the suspension with an 18G needle (5-10 times). The lysate was spun for 10 minutes and then centrifuged for 10 minutes at maximum speed (13K rpm) in a microcentrifuge. The pellet was discarded and the supernatant was then assayed with the Bradford protein assay kit (Biorad).

[209] The lysate (2 μl) was pipetted directly onto a dry 0.2 μm nitrocellulose membrane. The spot was air dried for about 30 seconds. The membrane was chopped so that each contained one spot. The spots were divided into pronase (1 mg / ml enzyme, 50 mM Tris, pH 7.5, 5 mM CaCl ₂ ), proteinase K (1 mg / ml enzyme, 50 mM Tris, pH 7.5, 5 mM CaCl ₂ ), neuraminidase ( At 37 ° C. in the presence of 20 mU / ml enzyme, 50 mM sodium acetate, pH 7.5, 5 mM CaCl ₂ , 100 μg / ml BSA), or periodic acid (20 mM, 0.5 M sodium acetate, pH 5) Incubated for 1 hour. Reagents were purchased from Roche (enzyme) and VWR (periodic acid). Membranes were washed in T-TBS wash buffer (0.1% Tween 20, 1 × TBS) (5 min) and in blocking buffer (3% BSA, T-TBS) for 2 hours at room temperature. Blocked and incubated overnight at 4 ° C. with 2 μg / ml primary antibody (ie DS6, CM1, C242, R24) in blocking buffer. The membrane was washed 3 times for 5 minutes in T-TBS and then in HRP conjugated goat anti-mouse (or human) IgG secondary antibody (Jackson Immunoresearch; diluted 1: 2000 in blocking buffer) at room temperature. Incubated for 1 hour. These immunoblots were washed three times and developed with an ECL system (Amersham).

[210] An immunoblot of the digested control lysate (Figure 2) shows that the CM1 signal was disrupted by proteolytic processing as expected for antibodies that recognize protein epitopes, but the glycolytic digest was not affected. Indicated. The C242 signal was destroyed by either proteolytic or glycolytic processing, as expected for antibodies that recognize glycotopes on proteins. The R24 signal that was not affected by proteolytic processing was destroyed by neuraminidase or periodic acid treatment, as expected for antibodies that recognize gangliosides. DS6 immunoblot of the dot blot of digested Caov-3 lysate showed loss of signal upon treatment with both proteolytic and glycolytic compounds. Thus, like C242, DS6 binds to a carbohydrate epitope on the protein core. In addition, the DS6 immunoblot signal was sensitive to neuraminidase treatment. Thus, like CanAg, CA6 is a sialic acid dependent glycotope.

[211] To confirm the carbohydrate character of CA6, Caov-3 lysate was spotted on a PVDF membrane and treated with the chemical deglycosyl trifluoromethanesulfonic acid (TFMSA) under nitrogen at ambient temperature for 5 minutes. The blot was washed with T-TBS and immunoblotted with CM1 or DS6 (FIG. 3). The DS6 signal was destroyed upon acid treatment, providing further evidence that CA6 is a glycotope. The enhanced CM1 signal upon TFMSA treatment indicates that this acid treatment does not affect the protein on the filter and suggests that the glycolytic treatment exposed the protein epitope recognized by CM1.

[212] To further elucidate the structure of carbohydrates in which CA6 is present, dot blots were digested with N-glycanase, O-glycanase and / or sialidase (FIG. 4). Caov-3 cell lysate (100 μg, 30 μl) was incubated with 2.5 μl of denaturing buffer (Glyko) containing SDS and β-mercaptoethanol for 5 minutes at 100 ° C. The denatured lysate was then digested with 1 μl N-glycanase, O-glycanase and / or sialidase A (Glyko) for 1 hour at 37 ° C. The digested lysate was then spotted on nitrocellulose (2 μl) and immunoblotted as before.

[213] N-glycanase had no apparent effect on the DS6 immunoblot signal. However, samples digested with sialidase produced no signal. Since O-glycanase could not digest sialylated O-linked carbohydrate without pretreatment with sialidase, there was no effect on the DS6 signal of samples treated with O-glycanase alone. In contrast, N-glycanase does not require pretreatment with glycoside enzymes for activity. The fact that N-glycanase treatment does not affect the DS6 signal suggests that the CA6 epitope is most likely present on the sialylated O-linked carbohydrate chain.

Example 3: Elucidation of an antigen having a CA6 epitope
[214] DS6 immunoprecipitates were analyzed by SDS-PAGE and Western blotting to identify antigens in which the CA6 sialoglycotope is present. Cell lysate supernatant (1 mL / sample; 3-5 mg protein) was pre-clarified with protein G beads (30 μl) equilibrated with 1 ml RIPA buffer while rotating at 4 ° C. for 1-2 hours. . All subsequent steps were performed on ice and / or in a cold room at 4 ° C. The pre-clarified beads were centrifuged in a microcentrifuge for a short time (2 to 3 seconds). The pre-clarified supernatant was transferred to a new tube and incubated overnight with rotation with 2 μg DS6. New equilibrated protein G beads (30 μl) were added to the lysate and incubated for 1 hour with rotation. The bead-lysate suspension was briefly centrifuged in a microcentrifuge and a sample of the lysate was collected after this immunoprecipitation, if desired. The beads were washed 5-10 times with 1 ml RIPA buffer.

[215] The immunoprecipitated DS6 sample was then treated with 30 μl neuraminidase (20 mU neuraminidase (Roche), 50 mM sodium acetate, pH 5, 5 mM CaCl _2.
, 100 μg / ml BSA) or 30 μl periodic acid (20 mM periodic acid (VWR), 0.5 M sodium acetate, pH 5) at 37 ° C. for 1 hour. They were then resuspended in 30 μl of 2 × sample loading buffer (containing β-mercaptoethanol). The beads were boiled for 5 minutes and the loading buffer supernatant was loaded onto a 4-12% or 4-20% Tris-Glycine gel (Invitrogen). The gel was developed in Laemli electrophoresis buffer at 125v for 1.5 hours. The gel sample was transferred onto a 0.2 μm nitrocellulose membrane (Invitrogen) overnight at 20 mA with a Mini Trans blot transfer device (Biorad). Membranes were immunoblotted with DS6 as described in Example 2 above.

[216] Alternatively, the immunoprecipitated beads are first denatured and then N-glycanase, O
-Enzymatic digestion with glycanase and / or sialidase A (Glyko). Resuspend the beads in 27 μl incubation buffer and 2 μl denaturing solution (Glyko)
Incubated at 100 ° C. for 5 minutes. After cooling to room temperature, detergent solution (2 μl) was added and the samples were incubated with 1 μl N-glycanase, O-glycanase and / or sialidase A for 4 hours at 37 ° C. After adding 5 × sample loading buffer (7 μl), the sample was boiled for 5 minutes. Samples were treated with SDS-PAGE as described above and immunoblotted.

[217] DS6 immunoprecipitates a> 250 kDa protein band found in antigen-positive cell lysates (FIGS. 5A, B and C). Some cell lines (ie T-47D) have doublets. This> 250 kDa band was disrupted in Caov-3 immunoprecipitates treated with neuraminidase or periodic acid (FIGS. 5A and B), suggesting that the CA6 epitope is on this> 250 kDa band. To do. The> 250 kDa band was also shown to be insensitive to N-glycanase treatment of immunoprecipitates, consistent with CA6 being on O-linked carbohydrate (FIG. 5F). Further supporting that the 250 kDa band is the CA6 antigen is the fact that DS6 does not immunoprecipitate such bands from cells negative for the DS6 antigen (FIGS. 5D and E).

[218] Several lines of evidence suggested that the CA6 antigen is Mucl. The CA6 antigen appeared to be a mucin because of its high molecular weight and sensitivity to O-linked carbohydrate specific glycolytic enzymes. Mucin overexpression has been elucidated, especially in breast and ovarian tumors, consistent with the main tumor reactivity of DS6. Furthermore, CA6 is not as sensitive to periodate precipitation as CanAg (sialoglycotopes on Muc1), suggesting that the CA6 antigen is highly O-glycosylated. The finding that DS6 immunoprecipitated> 250 kDa doublets in certain DS6-expressing cell lines suggested that CA6 was Muc1. A characteristic of human Muc1 is that two Muc1 proteins with different molecular weights are produced by the presence of two Muc1 alleles with different numbers of tandem repeats.

[219] DS from Caov-3 to see if CA6 is on Muc1
Six immunoprecipitates were treated with SDS-PAGE and immunoblotted with DS6 or Muc1 VNTR antibody CM1. As can be seen from FIG. 6A, CM1 reacts strongly with the> 250 kDa band immunoprecipitated by DS6. In FIG. 6B, DS6 and CM1 immunoprecipitates from HeLa cell lysates show the same> 250 kDa doublet when immunoprecipitated with either DS6 or CM1. These results indicate that the CA6 epitope is indeed on the Muc-1 protein. The DS6 doublet found in HeLa (and T-47D) cells can be explained by the fact that Muc-1 expression is governed by distinct alleles that differ in the number of tandem repeats.

[220] CM1 and DS6 bind to the same Muc-1 protein but different epitopes. Chemical deglycosylation of dot blots of Caov-3 lysate with trifluoromethanesulfonic acid (TFMSA) disrupted the DS6 signal (FIG. 3). However, this same process enhanced the CM1 signal. Deglycosylation may have exposed a hidden epitope for the CM1 antibody. Furthermore, comparing the results of flow cytometric binding of DS6 and CM1 (Table 4) demonstrates that the CA6 epitope is not present on all cells that express Mucl. It is interesting to note that the CA6 epitope is not present in the cell line Colo205 (Table 3), which is known to express high concentrations of Mucl CanAg sialoglycotopes.

^* MMF = maximum average relative fluorescence
Example 4: Quantitative analysis of released CA6 epitope
[221] Because the CA6 epitope is present on the molecule Muc1, which is known to be released into the bloodstream in many cancer patients, to determine whether such concentrations interfere with DS6 antibody therapy The quantitative operation was performed. When the circulating antibody binds to the antigen, it is thought that the immune complex is rapidly cleared from the blood. If a significant portion of the antibody dose is rapidly cleared from the circulation, the amount that reaches the tumor is reduced and the anti-tumor activity of the antibody therapeutic may be reduced. When the antibody is conjugated to a highly effective cytotoxic compound, rapid clearance of the conjugate may increase nonspecific toxicity. Thus, in the case of antibody-small molecule drug conjugates, such as DS6-DM1, it was expected that high concentrations of released antigen could reduce antitumor effects and increase dose limiting toxicity.

[222] Recent clinical trials of antibody therapeutics have provided information on the effect of released antigen concentration on pharmacokinetics. For example, in clinical trials using the antibody trastuzumab (Herceptin) used in the treatment of metastatic breast cancer expressing her2 / neu,
It has been shown that the pharmacokinetics of trastuzumab clearance does not change when the er2 / neu concentration is less than 500 ng / mL (Pegram et al., J. Clin. Oncol. 16 (8): 2659-71 (1998) ). Assuming that the molecular weight of the released Her2 / neu is 110,000 daltons, a molar concentration of released Her2 / neu of less than 4.5 nM appears to have little effect on pharmacokinetics.

  [223] In another example, clinical trials with cantuzumab mertansine (huC242-DM1) showed no correlation between the pretreatment release CanAg (C242 epitope) concentration and antibody clearance pharmacokinetics (Tolcher et al., J. Clin. Oncol. 21 (2): 211-22 (2003)). Similar to the CA6 epitope recognized by DS6, the CanAg epitope is a unique tumor-specific O-linked sialoglycotope on Muc1. However, due to the heterogeneity of the CanAg epitope, it is difficult to quantify in molar quantities. In the general population, the length of the Mucl allele depends on the number of tandem repeats of the variable number tandem repeat (VNTR) domain. There are several sites for O-linked glycosylation in each tandem repeat. In addition to the complexity of CanAg expression, the intrinsic glycosyltransferase activity varies from cell to cell. Thus, there is a wide range of CanAg epitopes per Muc molecule even in a single patient. Furthermore, the ratio of CanAg epitopes per Muc molecule will vary across patient populations. For this, the CanAg released into the serum sample is measured by sandwich ELISA. In this case, released Mucl with the CanAg epitope is captured by C242 and detected by the biotinylated C242 / streptavidin HRP system. The released CanAg is quantified in standard units (U) proportional to the number of epitopes per ml of serum rather than the molar concentration of Mucl. Similarly, a similar situation arises for the quantification of released CA6 epitope. In contrast, trastuzumab has only one epitope per released her2 / neu molecule, greatly quantifying the released antigen.

[224] To examine the correlation between the released CA6 epitope concentration and that seen in clinical trials with trastuzumab and cantuzumab-mertansine, the molar concentration of complex released epitopes such as sialoglycotopes on Muc1 Developed a method for seeking. First, a simple sandwich ELISA assay for DS6 was established. The concept of this assay is shown in FIG. 7A. DS6 was used to capture Muc1 with the CA6 epitope. Since each Mucl molecule has multiple CA6 epitopes, biotinylated DS6 was also used as a tracer antibody. Biotinylated DS6 bound to captured CA6 was detected by streptavidin-HRP using ABTS as a substrate. CA6 epitopes were captured from sera of ovarian cancer patients or from standards obtained from a commercial Muc1 test kit (CA15-3) used to monitor released Muc1 in breast cancer patients. Unit of DS6 /
ml was arbitrarily set equal to the unit of CA15-3 standard / ml.

FIG. 7B shows the results of DS6 sandwich ELISA using CA15-3 standard. The resulting curve is very similar to that obtained with the CA15-3 standard in the CA15-3 assay. In order to convert DS6 units / ml to CA6 molarity, a biotinylated DS6 standard curve is needed to convert the signal to DS6 picograms. Assuming that the stoichiometric amount of CA6 epitope and biotinylated DS6 antibody is 1: 1 and the molecular weight of biotinylated DS6 is 160,000 daltons, the number of moles of CA6 captured per sample addition volume can be calculated. it can.

[226] FIGS. 8A and B represent two alternative methods for generating a standard curve of biotinylated DS6. In FIG. 8A, biotinylated DS6 is captured using a goat anti-mouse IgG polyclonal antibody and detected in the same manner as used for the sandwich ELISA shown in FIG. In the method shown in FIG. 8B, biotinylated DS6 is placed directly on the ELISA plate and detected as in FIG. 8A. As seen in FIG. 8C, the biotinylated DS6 standard curve generated by each method is in good agreement.

[227] Table 5 shows the analysis of serum samples from ovarian cancer patients for various released antigens. In general, treatment of ovarian cancer patients is monitored by measuring units / ml of released CA125 using a CA125 ELISA. CA125 status was determined for serum samples. Breast cancer patients are typically monitored using a CA15-3 ELISA and measuring units / ml of released Muc1 utilizing capture and detection of antibodies that recognize different epitopes than those recognized by DS6. In Table 5, CA15-3 is measured in serum samples of ovarian cancer patients.

¹ measured by a commercially available ELISA kit
² commercial CA15-3 measured by standard (1 CA15-3 U = 1 DS6 U )
³ goat anti-mouse IgG and biotin-DS6 standard curve
⁴ Biotin-DS6 standard curve
[228] For the CA15-3 values reported in Table 5, a CA15-3 enzyme immunoassay kit commercially available from CanAg Diagnostics was used. For DS6 units / ml, a standard curve was generated using a CA15-3 standard (from a CA15-3 enzyme immunoassay kit commercially available from CanAg Diagnostics) in a DS6 sandwich ELISA. DS6 units / ml were arbitrarily set equal to CA15-3 units / ml. In the last two columns, the picomolar concentration (pM) of released CA6 was calculated using the standard curve of biotinylated DS6 shown in FIG. 8C.

[229] For quantitative analysis of CanAg concentrations, CanAg serum concentrations were reported before treatment for patients participating in the Kanzumab Mertansin clinical trial (Tolcher et al., J. Clin. Oncol. 21 (2 ): 211-22 (2003)). A CanAg standard curve was generated using a CanAg standard by an ELISA assay similar to that described for DS6. C242 was used to capture the CanAg standard. Detection of captured CanAg was achieved using a biotinylated C242 tracer followed by development with streptavidin-HRP using ABTS as a substrate. A biotinylated-C242 standard curve was generated as was done for biotinylated DS6 and the units / ml of circulating CanAg epitope could be converted to molar concentrations. Table 6 reports the CanAg concentration and the corresponding calculated circulating CanAg molar concentration from the Kanzumab Mertansin clinical trial patients.

Pretreatment concentration of circulating CanAg measured by ¹ sandwich ELISA
^Two goat anti-mouse IgG and biotin-C242 standard curves
⁴ Biotin-C242 standard curve
[230] Comparing the pM concentration of released CA6 in ovarian cancer patients with that calculated for released CanAg in CanAg positive cancer patients, it is generally shown that the released CA6 concentration is similar to the released CanAg concentration. In addition, only 2 out of serum samples 16 from ovarian cancer patients have concentrations higher than 4.5 nM (serum samples 5 and 9 whose signal is outside the standard curve range); this is Her2 / neu positive above this This is the concentration at which herceptin pharmacokinetics changed in clinical trials for breast cancer patients. CanAg concentrations higher than 4.5 nM were found in only 3 of 37 clinical trial patients. In this clinical trial, there was no correlation between the released CanAg concentration and the more rapid cantizumab meltansin clearance. However, patients with the highest CanAg concentration (31240 U / ml) sampled only 8 hours after infusion. These results indicate that certain epitopes of Mucl, such as CA6 and CanAg, are released in cancer patients, but not at concentrations that interfere with antibody therapy treatment.

Example 6: Cloning of murine DS6 antibody variable region
[231] Murine monoclonal antibodies, such as DS6, are recognized as foreign by the human immune system and have limited utility in clinical settings. Patients rapidly express human anti-murine antibodies (HAMA) so that murine antibodies are cleared rapidly. For this reason, a humanized DS6 (huDS6) antibody was prepared by resurfacing the variable region of murine DS6 (muDS6).

[232] The murine DS6 antibody variable region was cloned by RT-PCR. Total RNA was purified from DS6 hybridoma cells in a close-packed T175 flask using the Quiagen RNeasy miniprep kit. The RNA concentration was measured by UV spectrophotometry, and RT reaction was performed on 4-5 μg of total RNA using Gibco Superscript II kit and random tetramer primers.

[233] The PCR reaction was performed according to the method described in Wang Z et al., J. Immunol Methods, January 13; 233 (1-2): 167-77 (2000) using degenerate primers. The RT reaction mix was used as it was for the degenerate PCR reaction. 3 'side L primer, HindKL

And 3 'heavy chain primer, BamIgG1

PCR primers for the 5 'end are Sac1MK for the L chain

For the heavy chain, EcoR1MH1

And EcoR1MH2

(Mixed base: H = A + T + C, S = G + C, Y = C + T, K = G
+ T, M = A + C, R = A + G, W = A + T, V = A + C + G, N = A + T + G + C).
[234] The PCR reaction was standard except that it was supplemented with 10% DMSO (50 μl of reaction mix was prepared with 1 × final concentration of 1 × reaction buffer (ROCHE), 2 mM dNTP, 1 m each)
M primer, 2 μl RT reaction, 5 μl DMSO, and 0.5 μl Taq (ROCHE)). PCR using a program applied by MJ research thermocycler from Wang Z et al., (J. Immunol Methods, 13 January; 233 (1-2): 167-77 (2000))
The reaction was carried out: 1) 94 ° C., 3 minutes; 2) 94 ° C., 15 seconds; 3) 45 ° C., 1 minute; 4) 72 ° C., 2 minutes; 5) 29 times back to step 2; Extension process 72 ° C, finished in 10 minutes. The PCR product generated with the PCR primers was cloned into pBluescript II SK + (Stratagene) with restriction enzymes. The heavy and light chain clones were sequenced by the Seqwright sequencing service.

[235] Further PCR and cloning were performed to confirm the 5 'terminal cDNA sequence. DS6 L chain and H chain cDNA sequences determined from degenerate PCR clones were input into the NCBI Blast search feb site, and the murine antibody sequences with the signal sequences presented were stored. PCR primers were designed from these signal peptides using extension sequences conserved between related DNA sequences. EcoR1 restriction sites were added to the leader sequence primers (Table 7) and used in the RT-PCR reaction described above.

[236] In order to identify and avoid possible polymerase-generated sequence errors, the sequences of several individual light and heavy chain clones were determined. Only one sequence was obtained for both light and heavy chain RT-PCR clones. These sequences were sufficient to design primers that could amplify the murine DS6 light and heavy chain sequences and extend them to the signal sequence. Subsequent clones obtained from these follow-up PCR reactions confirmed the 5 'end sequence of the variable region changed by the first degenerate primer. The cumulative results from the various cDNA clones resulted in the final murine DS6 light and heavy chain sequences shown in FIG. Using the Kabat and AbM definitions, three light and heavy chain CDRs were identified (Figures 9 and 10). By searching the NCBI IgBlast database, the mu chain antibody L chain variable region is murine IgV. It was most likely derived from the ap4 germline gene, indicating that the heavy chain variable region was most likely derived from the murine IgVh J558.41 germline gene (FIG. 11).

Example 7: Determination of variable region surface residues of DS6 antibody
[237] The antibody resurfacing method described by Pedersen et al. (1994) and Roguska et al. (1996) begins by estimating the surface residues of murine antibody variable sequences. Surface residues are defined as amino acids that allow water molecules to reach at least 30% of their total surface area. Since the structure for finding the surface residues of muDS6 was undecided, we aligned 10 antibodies with the most homologous sequences in 127 antibody structure files (FIG. 12). For these aligned sequences, solvent accessibility for each Kabat position was averaged (FIGS. 13A and B).

[238] A second round of analysis was performed by comparison of antibody subsets containing two identical residues flanking on both sides for a surface location with average accessibility of 25-35% (FIGS. 13A and B). After the second round of analysis, 21 putative surface residues for the muDS6 heavy chain were increased to 23, and Tyr3 and Lys23 were added to the list of residues with a putative surface accessibility higher than 30%. Although the Kabat definition of H chain CDR1 is used in most antibodies resurfaced by the present inventors, the Ab chain definition was used unintentionally in the calculation for DS6, so the H chain residue T28 is the same as in other cases. Not defined as framework surface residues. The number of light chain surface positions was reduced from 16 to 15 as the surface accessibility of the second round of analysis Ala80 was reduced from 30.5% to 27.8%. Together, the muDS6 heavy and light chain variable region sequences have 38 putative surface accessible framework residues.

Example 8: Selection of human antibodies
[239] The surface position of the murine DS6 variable region was compared with the corresponding position of the human antibody sequence in the Kabat database (Johnson G., Wu TT. Nucleic Acids Res. 1 January; 29 (1): 205-6 (2001)). Surface residues were extracted and aligned from natural heavy and light chain human antibody pairs using antibody database processing software (Searle, 1998). In particular, the human antibody variable region surface with the most identical surface residue was chosen to replace the murine DS6 antibody variable region surface residue, taking into account the position within 5 cm of the CDR.

Example 9: Expression vectors for chimeric and humanized antibodies
[240] The heavy and light chain pairing sequences were cloned into a single mammalian expression vector. A PCR primer for the human variable sequence created a restriction site where a human signal sequence could be added into the pBluescript II cloning vector. These variable sequences were then cloned into mammalian expression plasmids using EcoR1 and BsiWI or HindIII and ApaI for the L or H chain, respectively (FIG. 14). The light chain variable sequence was cloned into the human IgKappa constant region with the reading frame matched, and the heavy chain variable sequence was cloned into the human IgGamma1 constant region sequence. In the final expression plasmid, the human CMV promoter induces expression of both light and heavy chain cDNA sequences.

Example 10: Identification of residues that may negatively affect DS6 activity
[241] Currently, a molecular model of the antibody of interest is constructed to identify residues that are close to the CDR as residues that may be problematic in most humanizations. Since the number of resurfaced antibodies is increasing and working from historical experience in assuming the problem is at least as effective as model building, no molecular model was built for DS6. Instead, murine DS6 surface residues were compared to those of conventionally resurfaced antibodies and identified from low to high risk residues for effects on antibody binding activity.

[242] In both elucidated antibody structures and molecular models available from conventional humanization, repeated identifications of similar residue sets within 5 か ら of the CDRs are identified. Using this data, murine DS6 residues that appear to be in close proximity to the CDRs, and possibly within 5 ', are listed in Table 1. Many of these positions were also exchanged during conventional humanization, but only heavy chain position 74 has so far lost binding activity. In order to preserve the binding activity of the murine antibody, the murine residue at this position was retained in both huC242 and huB4. On the other hand, in humanized 6.2G5C6, the activity was not lost when this same position was replaced with the corresponding human residue (6.2G5C6 is an anti-IGF1-R antibody, often simply referred to as anti-C6). . While any of the residues in Table 1 can pose problems in humanized antibodies, the heavy chain residue P73 is particularly problematic due to prior experience at this position.

Example 11: Selection of the most homologous human surface
[243] The surface of a human antibody that is a candidate for resurfacing muDS6 is treated with Kab
It was determined from the at antibody sequence database using SR software. This software provides an interface for searching only specific residue positions against the antibody database. In order to preserve the natural pair, both light chain and heavy chain surface residues were compared to each other. To rank the sequence identity, the most homologous human surface from the Kabat database was aligned. The top three surfaces aligned by SR Kabat database software are shown in Table 2. These surfaces were then compared to identify which human surface required the least exchange to make the residues identified in Table 1. Anti-Rh (D) antibody 28E4 requires the least number of surface residue exchanges (11 in total) and only three of these are included in the list of potentially problematic residues. The 28E4 antibody is the best candidate for resurfacing muDS6 because it provides the most homologous human surface.

Example 12: Construction of DNA sequence of humanized DS6 antibody
[244] The 11 surface residues were exchanged for those of DS6 by PCR mutagenesis. PCR mutagenesis was performed on murine DS6 variable region cDNA clones in order to construct the resurfaced human DS6 gene. In order to generate amino acid changes necessary for resurfaced DS6, the primer set for humanization shown in Table 8 below was designed.

[245] The PCR reaction was standard except that it was supplemented with 10% DMSO (50 μl of reaction mix was prepared with 1 × final concentration of 1 × reaction buffer (ROCHE), 2 mM dNTP, 1 m each)
M primer, 100 ng template, 5 μl DMSO, and 0.5 μl Taq (ROCHE)). The MJ research thermocycler was operated using the following program: 1) 94 ° C, 3 minutes; 2) 94 ° C, 15 seconds; 3) 55 ° C, 1 minute; 4) 72 ° C, 1 minute; 29); 6) Final extension step 72 ° C., finished in 4 minutes. PCR products were digested with their corresponding restriction enzymes and cloned into the pBluescript cloning vector. To confirm amino acid changes, the clones were sequenced.

[246] Since the exchange of heavy chain residue P73 caused problems in the past, two versions of heavy chain were constructed: one containing human 28E4 T73 and one carrying murine P73. In both versions of humanized DS6, the other 10 surface residues were exchanged from murine to human 28E4 residues (Table 2). According to normal nomenclature, the closest human being is version 1.0. This is because it has all 11 human surface residues. If more versions are needed, the heavy chain version retaining murine P73 is named version 1.2, leaving version 1.1 for the version containing the maximum number of murine residues. The amino acid sequences of the two humanized versions are shown in FIGS. 15A and B, aligned with the murine DS6 amino acid sequence. Both humanized DS6 antibody genes were cloned into antibody expression plasmids (Figure 14) for transient and stable transfection. The cDNA and amino acid sequences of the light chain variable region of humanized versions v1.0 and v1.2 are identical and are shown in FIG. The heavy chain cDNA and amino acid sequences of humanized versions v1.0 and v1.2 are shown in FIGS.

Example 13: Expression and purification of huDS6 in CHO cells and affinity measurement
[247] In order to determine whether the humanized version retained the binding affinity of muDS6, the antibody had to be expressed and purified. CHO cells were transfected with each antibody expression plasmid. Since the transient expression level of huDS6 was very low, a stable cell line was selected.

[248] CHODG44 cells (4.32 × 10 ⁶ cells / plate) in non-selective medium (Alpha MEM + nucleotide (Gibco); 4 mM L-glutamine, 50 U / ml penicillin, 50 μg / ml streptomycin in 15 cm plates And supplemented with 10% v / v FBS) and placed in a humidified incubator at 37 ° C., 5% CO ₂ . The next day, cells were transfected with huDS6 v1.0 and v1.2 expression plasmids using a modified Quiagen recommended protocol for Polyfect transfection. Non-selective medium was aspirated from the cells. Plates were washed with 7 ml preheated (37 ° C.) PBS and supplemented with 20 ml non-selective medium. Plasmid DNA (11 μg) was diluted into 800 μl of hybridoma SFM (Gibco). Then 70 μl of Poly
Fect (Quiagen) was added to the DNA / SFM mixture. Then Polyfect
The mixture was gently vortexed for a few seconds and incubated at ambient temperature for 10 minutes. Non-selective medium (2.7 ml) was added to the mixture. The final mixture was incubated with the seeded cells for 24 hours.

[249] The transfection mixture / medium was removed from the plate and the cells were then trypsinized and counted. Cells were then loaded with selective medium (Alpha MEM-nucleotide; 4 mM L-glutamine, 50 U / ml penicillin, 50 μg / ml streptomycin, 10% v / v FBS, 1.25 mg / ml G418 in a 96-well plate. Supplements) (250 μl / well) were inoculated at various densities (1800, 600, 200, and 67 / well). Cells were incubated for 2-3 weeks, supplemented with media if necessary. Wells were screened for antibody production levels by quantitative ELISA. Immunolon 2HB 96-well plate was coated with goat anti-human IgG F (ab) ₂ antibody (Jackson Immunoresearch; 1 μg / well in 100 μl of 50 mM sodium carbonate buffer (pH 9.6)) and 1. Incubated for 5 hours. All subsequent steps were performed at ambient temperature. The wells were washed twice with T-TBS (0.1% Tween 20, TBS) and blocked with 200 μl blocking buffer (1% BSA, T-TBS) for 1 hour. Wells were washed twice with T-TBS. In a separate plate, antibody standard EM164 (100 ng / ml) and culture supernatant were serially diluted with blocking buffer (1: 2 or 1: 3). These dilutions (100 μl) were transferred to ELISA plates and incubated for 1 hour. Wells were washed 3 times with T-TBS and goat anti-human IgG Fc-AP (Jackson Immunoresear diluted 1: 3000 in blocking buffer).
ch) Incubated with 100 μl for 45 minutes. The wells were washed 5 times with T-TBS and then 100 μl of PNPP developer (10 mg / ml PNPP (p-nitrophenyl phosphate disodium salt; Pierce), 0.1 M diethanolamine, pH 10.3 buffer) For 25 minutes. Absorbance at 405 nm was measured with an ELISA plate reader. The antibody concentration was determined using the absorbance reading (of the culture supernatant) in the linear portion of the standard curve.

[250] The highest producing clones identified by ELISA were then expanded to prepare frozen cell stocks. In order to produce sufficient quantities to purify, cells were spread on 15 cm plates with 30 ml selective medium (approximately 1 × 10 ⁶ cells / plate) and incubated for 1 week. The culture supernatant was collected into a 250 ml conical tube, centrifuged in a tabletop centrifuge (2000 rpm, 5 minutes, 4 ° C.), and then aseptically filtered through a 0.2 μm filtration device.

[251] To purify DS6, NaOH pellets were added to the filtered culture supernatant to a final pH of 8.0. A Hi Trap rProtein A column (Amersham) was equilibrated with 20-50 column volumes of binding buffer. The supernatant was loaded onto the column with a peristaltic pump. The column was then washed with 50 column volumes of binding buffer. Bound antibody was eluted with elution buffer (100 Mm acetic acid, 50 mM NaCl, pH 3) into a test tube attached to the fraction collector. The eluted antibody was then neutralized with neutralization buffer (2M K ₂ HPO ₄ , pH 10.0) and then dialyzed overnight in PBS. The dialyzed antibody was filtered through a 0.2 μm syringe filter. Absorbance at 280 nm was measured to determine the final protein concentration.

[252] The affinity of purified huIgG was compared with muDS6 by flow cytometry. In the first set of experiments, direct binding to the CA6-expressing cell line WISH was measured. As shown in FIG. 18A, chimeric DS6, huDS6 v1.0, and huDS6 v1.2 show very similar affinities with an apparent KD of 3.15 nM, 3.71 nM, and 4.2 nM, respectively; , Suggesting that resurfacing did not perturb the CDRs. These affinities are only slightly lower than those of muDS6 shown in FIG. 18B (Kd = 1.93 nM).

To confirm that each version of huDS6 retains muDS6 affinity, comparative binding experiments were performed. The advantage of this format is that the same detection system is used for both murine and human antibodies, namely biotin-muDS6 / streptavidin-DTAF. Due to the second reagent used for indirect detection of binding, the experimental measurement of apparent Kd may fluctuate in some cases. By comparing the apparent Kd of biotin-muDS6 with goat anti-mouse FITC (Kd = 2.80 nM; FIG. 18B) or with streptavidin-DTAF (Kd = 6.76 nM; FIG. 19A), This will be explained. The results of a competitive binding assay comparing the ability of muDS6, huDS6 v1.0, and huDS6 v1.2 to compete with biotin-muDS6 are shown in FIG. 19B. The apparent EC ₅₀ is 12.18 nM, 37.07 nM, and 22.64 nM for muDS6, huDS6 v1.0, and huDS6 v1.2, respectively. These results indicate that muDS6 resurfacing to make humanized DS6 hardly reduces binding affinity.

Example 14: Preparation of DS6-DM1 cytotoxic conjugate
[253] DS6 antibody (8 mg / ml) was added in an 8-fold molar excess of N-succinimidyl-4- (
Modification with 2-pyridyldithio) pentanoate (SPP) introduced a dithiopyridyl group. Reactions were performed in 95% v / v Buffer A (50 mM KPi, 50 mM NaCl, 2 mM EDTA, pH 6.5) and 5% v / v DMA for 2 hours at room temperature. The slightly swollen reaction mixture was gel filtered through a NAP or Sephadex G25 column (equilibrated in buffer A). The degree of modification was determined by measuring the absorbance of the antibody at 280 nm and the absorbance of DTT released 2-mercaptopyridine (Spy) at 280 and 343 nm. The antibody 2.5 mg / mL modified DS6 was then used with a 1.7-fold molar excess of N ^{2 ′} -deacetyl-N ^{2 ′} -(3-mercapto-1-oxopropyl) -maytansine (L-DM1) over Spy. And conjugated. This reaction was performed in buffer A (97% v / v) with DMA (3% v / v). The reaction was incubated at room temperature overnight for about 20 hours. The opaque reaction mixture was centrifuged (1162 × g, 10 min) and then NAP-25 or S300 (Tandem 3, 3 × 26/10 desalted) equilibrated in buffer B (1 × PBS, pH 6.5) Column, G25 medium) The supernatant was gel filtered through a column. The pellet was discarded. The conjugate was aseptically filtered through a 0.22 μm Millex-GV filter and dialyzed in buffer B containing Slide-A-Lyser. The number of DM1 molecules linked per molecule of DS6 was determined by measuring the absorbance of the filtered material at both 252 nm and 280 nm. The DM1 / antibody ratio was found to be 4.36, and the process yield of conjugate DS6 was 55%. The concentration of the conjugated antibody was 1.32 mg / mL. This purified conjugate was biochemically resolved by size exclusion chromatography (SEC) and 92% was found to be monomeric. Analysis of DM1 in the purified conjugate showed that 99% was covalently bound to the antibody. FIG. 20 shows that flow cytometric binding of DS6-DM1 conjugate and unmodified DS6 to Cao-3 cells only slightly reduced affinity due to conjugation of DS6.

Example 15: In vitro cytotoxicity of DS6-DM1
[254] DS6 as a naked antibody did not show proliferation or growth inhibitory activity in cell culture (FIG. 21). However, when DS6 is incubated with cells in the presence of DM1 conjugates to goat anti-human IgG heavy and light chains, DS6 effectively targets and delivers this conjugate to the cells resulting in indirect cytotoxicity. (FIG. 21). To further test the intrinsic activity of naked DS6, complement dependent cytotoxicity (CDC) assays using murine and humanized DS6 were performed. HPAC and ZR-75-1 cells (25000 cells / well) were cultured in 96-well plates in the presence of 5% human or rabbit serum and various dilutions of murine or human DS6 in 200 μl RHBP medium (RPMI- 1640; 0.1% BSA, 20 mM HEPES (pH 7.2-7.4), 100 U / ml penicillin, and 100 ug / ml streptomycin). Cells were incubated for 2 hours at 37 ° C. Alamar Blue (final concentration 10%) reagent (Biosource) was then added to the supernatant. 5-2 cells
After incubation for 4 hours, fluorescence was measured. Both murine and humanized DS6 did not affect the complement dependent cytotoxicity (CDC) assay (FIG. 22). This suggests that conjugation to toxic effector molecules is required for DS6 to be used in therapy.

[255] Cytotoxicity of DS6 antibodies conjugated to maytansinoids was examined using two different assay formats in various DS6-positive cell lines. Colony formation assays were performed on 6-well plates by seeding cells (1000-2500 cells / well) in 2 ml of conjugate diluted in medium. Cells were exposed continuously to several concentrations of conjugate, typically 3 × 10 ^{−11 to} 3 × 10 ⁻⁹ M, and incubated for 5-9 days in a humidified chamber at 37 ° C., 6% CO ₂ . Wells were washed with PBS and colonies were stained with 1% w / v crystal violet / 10% v / v formaldehyde / PBS solution. The unbound dye was then thoroughly washed from the wells with distilled water and the plates were allowed to air dry. Colonies were counted with a Leica StereoZoom 4 dissecting microscope.

[256] Colony formation rate (PE) was calculated as number of colonies / number of inoculated cells. Viability was calculated as PE of treated cells / PE of untreated cells. The IC ₅₀ concentration was determined by graphing cell viability versus the molar concentration of the conjugate. In the colony formation assay (FIG. 23), DS6-DM1 was effective in killing Cao-3 cells with an estimated IC ₅₀ of 800 pM. Antigen-negative cell A375 was only slightly affected by the highest concentration of DS6-DM1 tested at 3 × 10 ⁻⁹ M conjugate; this indicates that the cytotoxic activity of the conjugate specifically affects antigen-expressing cells. Prove that it is directed. However, many other DS6 positive cell lines were not particularly sensitive to immunoconjugates despite being clearly sensitive to maytansine. Cervical cell lines (HeLa, KB, and WISH) were sensitive to the conjugates, but only a certain number of ovarian and breast cell lines showed a cytotoxic effect. The pancreatic cell line did not appear to be affected.

[257] In the MTT assay, cells were seeded in 96-well plates at a density of 1000-5000 cells / well. Cells were seeded in 200 μl medium with serial dilutions of naked DS6 or DS6-DM1 immunoconjugates. Samples were tested in triplicate. The cells and antibody / conjugate mixture were then incubated for 2-7 days before MTT ([3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide)] assay. Survival was assessed. MTT (50 μg / well) was added to the culture supernatant and incubated at 37 ° C. for 3-4 hours. The medium was removed and MTT formazan was dissolved in DMSO (175 μl / well). Absorbance at 540 to 545 nm was measured. In the MTT cell viability assay (FIG. 24C), the immunoconjugate was able to effectively kill Cao-3 cells with an estimated IC ₅₀ of 1.61 nM. Compared to the ineffective naked antibody, the well containing the highest concentration of conjugate contained no viable cells (FIGS. 21 and 24).

[258] MTT assay results for other cell lines were slightly different (FIG. 24A).
, B and D to I). In many cases, some cytotoxicity was seen, but the conjugate could not completely kill the entire cell population (except for WISH cells). BT-20, OVCAR5, and HPAC cells were particularly resistant: in wells with the highest concentration of conjugate (32 nM), more than 50% of the cells were still alive.

Example 16: In vivo anti-tumor activity of conjugates
[259] To demonstrate the in vivo activity of the DS6-DM1 conjugate, human tumor xenografts were established in SCID mice. A subcutaneous model of the human cervical cancer cell line KB was developed. KB cells were grown and harvested in vitro, and 5 × 10 ^{6 in} 100 μL of serum-free medium were injected under the right shoulder of each mouse and allowed to grow for 6 days to an average tumor volume of 144 ± 125 mm ^3. Started. Mice were intravenously administered daily for 6 days with PBS, DM1 150 μg / kg conjugate, or DM1 225 μg / kg conjugate (2 mice in each group). During the treatment period, the poisoning reaction was monitored once a day. During the treatment period, tumor volume (FIG. 25A) and corresponding body weight (FIG. 25B) were monitored.

[260] KB tumors treated with PBS control grew rapidly with a doubling time of about 4 days. In contrast, mice treated with the conjugate showed complete tumor regression in both groups; 14 and 18 days after initiation of treatment in the 225 μg / kg and 150 μg / kg groups, respectively. At the 150 μg / kg dose, tumor delay was about 70 days. The treatment at 225 μg / kg was cured because there was no evidence of tumor recurrence at the end of the study on day 120. As seen in FIG. 25B, mice in the 150 μg / kg group showed no weight loss; this indicated that this dose was well tolerated. At the higher dose, the mice only experienced a 3% temporary weight loss. During the 5 day treatment period, the mice showed no visible signs of poisoning. Taken together, this study demonstrates that DS6-DM1 treatment can cure mouse KB xenograft tumors at non-toxic doses.

[261] DS6-DM1 activity was further tested on a panel of subcutaneous xenograft models (
(See FIG. 26). The tumor cell line used to form the xenografts showed some in vitro maytansine sensitivity and CA6 epitope density (see Table 9 below). OVCAR5 cells and TOV-21G are ovarian tumor cell lines; HPAC is a pancreatic tumor cell line; HeLa is a cervical tumor cell line. OVCAR5 and TOV-21G cells show low surface CA6 expression; HeLa cells show intermediate levels of surface CA6 expression; HPAC shows high CA6 surface expression density. TOV-21G and HPAC cells are maytansine sensitive; OVCAR5 and HeLa cells are 2-7 times less sensitive to maytansine.

^* Average maximum relative average fluorescence
[262] The 4 cell lines described above were expanded in vitro, collected, and 1 × in 100 μL of serum-free medium.
10 ⁷ were injected under the right shoulder of each mouse (6 mice per model), allowed to grow for 6 days, and 57.6 ± 6.7 for the test and control groups, respectively, with an average tumor volume of OVCAR5 And 90.2 ± 13.4 mm ³ , 147 ± 29.6 and 176.2 ± 18.9 mm ^{3 for} the HPAC test group and control group, respectively, and 194.3 ± 37 for the HeLa test group and control group, respectively. .2 and 201.7 ± 71.7 mm ³ , and for TOV-21G test and control groups, 96.6 ± 22.8 and 155.6 ± 13.4 mm ³ , respectively. did. For each model, three control mice were treated intravenously twice weekly with PBS twice and three test mice with twice weekly conjugate (DM1 600 μg / kg). During the treatment period, the toxic response was monitored once a day, and the tumor volume and body weight were monitored during the treatment period. Conjugate effects for the various models are shown graphically in FIGS. 26A, C, E and G, and the corresponding body weights are plotted in FIGS. 26B, D, F and H. OVCAR5, TOV-21G and HPAC cell lines formed progressive tumors as seen in the PBS control of each model. The HeLa model had a lag phase of about 3 weeks before the start of logarithmic growth. In all models, DS6-DM1 conjugate treatment resulted in complete regression of tumors in all mice. For TOV-21G, HPAC, and HeLa models, mice remain tumor free on day 61. In the OVCAR5 model, tumors recurred approximately 45 days after tumor inoculation. Thus, in this model, DS6-DM1 treatment delays tumor growth for about 34 days. This growth delay is important because OVCAR5 cells are less sensitive to maytansine and have lower CA6 epitope expression. Tumor regression is more pronounced in models with higher CA6 epitope density or with higher maytansine sensitivity. It is important to note that only two doses were given. There was no weight loss, so the regimen adopted for this study was clearly not toxic to mice. It appears that healing can be achieved by administration of additional or higher amounts of the conjugate.

[263] Human ovarian cancer is mostly peritoneal disease. OVCAR5 cells, as an intraperitoneal (IP) model of SCID mice, progress and proliferate in the same manner as human disease to form tumor nodules and produce ascites. To demonstrate activity in the IP model, mice with OVCAR5 IP tumors were treated with DS6-DM1 (FIG. 27). OVCAR5 cells were grown in vitro, harvested, and injected intraperitoneally with 1 × 10 ⁷ cells in 100 μL of serum-free medium. Treatment began when tumors were allowed to grow for 6 days. Mice were treated with PBS or DM1 600 μg / kg DS6-DM1 conjugate once a week for 2 weeks to monitor weight loss caused by peritoneal disease. By day 28, mice in the PBS group had lost more than 20% and were euthanized. Treatment groups were euthanized on day 45 after weight loss exceeded 20%. This study demonstrates that DS6-DM1 can delay tumor growth in the advanced OVCAR5 model despite the fact that OVCAR5 is less sensitive to maytansine and has fewer CA6 epitopes per cell. Since the regimen adopted did not induce visible signs of poisoning, additional and higher doses could be used to achieve further tumor growth delay or healing.

Example 17: Synthesis and characterization of DS6-SPP-taxoid MM1-202 cytotoxic conjugate
[264] DS6 was modified with N-sulfosuccinimidyl 4-nitro-2-pyridyl-pentanoate (SSNPP) linker. To 50 mg DS6 antibody in 90% buffer A, 10 equivalents of SSNPP in DMA was added. The final concentration of antibody was 8 mg / ml. The reaction was stirred at room temperature for 4 hours and then purified by G25 chromatography. The degree of modification of the antibody was measured spectrophotometrically by absorbance at 280 nm (antibody) and 325 (linker) and was found to have linker 3.82 / antibody. The amount of recovered antibody was 43.3 mg, and a yield of 87% was obtained. A conjugate of DS6-nitro SPP was conjugated with taxoid MM1-202 (1812P.16). Conjugation was performed on a 42 mg scale in 90% Buffer A, 10% DM1. The taxoid was added 4 times at 0.43 equivalent / linker (each time) over about 20 hours. By this time, the reaction was noticeably cloudy. The product conjugate recovered in about 64% yield after G25 purification contained about 4.3 / antibody of taxoid, leaving about 1 equivalent of unreacted linker. To inactivate the unreacted linker, 1 equivalent of cysteine / unreacted linker was added to the conjugate with stirring overnight. When cysteine was added, a clear yellow coloration was observed, indicating the release of thiopyridine. The reaction solution was then dialyzed in buffer B / 0.01% Tween 20 followed by further dialyzing over several days in buffer B alone. The final conjugate contained drug 2.86 / antibody. The antibody recovery amount was 14.7 mg, and a yield of 35% was obtained as a whole. The conjugate was further biochemically resolved by SEC and found to contain 89% monomer, 10.5% dimer, and 0.5% higher molecular weight aggregate.

[265] FIG. 28 shows the results of flow cytometry analysis comparing the binding of DS6-SPP-taxoid MM1-202 and DS6 antibody to HeLa cells. This result indicates that DS6 retains binding activity when conjugated to a taxane.

[139] Other maytansines can be used in other embodiments of the present invention, including thiol- and disulfide-containing maytansinoids with mono- or di-alkyl substitution at the carbon atom bearing the sulfur atom. These include maytansinoids having the following: C-3, C-14 hydroxymethyl, C-15 hydroxy, or C-20 desmethyl, acylated amino acid side chains in which the acyl group carries a hindered sulfhydryl group Wherein the carbon atom of the acyl group carrying the thiol functional group has 1 or 2 substituents, the substituents being CH 3, C 2 H 5, linear or branched alkyl having 1 to 10 carbon atoms or Alkenyl, cyclic alkyl or alkenyl having 3 to 10 carbon atoms, phenyl, substituted phenyl, or heterocyclic aromatic or heterocyclic group, and one of the substituents may be H; Acyl groups have a straight chain of at least 3 carbon atoms in length between the carbonyl function and the sulfur atom.

[149] In a preferred embodiment of formula (VII), formula (VII) wherein R1 is methyl and R2 is H
VII).
[150] The maytansinoids described above can be conjugated to the anti-CA6 antibody DS6 or a homologue or fragment thereof. In that case, using a thiol- or disulfide functional group, C-14 hydroxymethyl, C-15 hydroxy, or C-20 desmethyl in the acyl group of the acylated amino acid side chain in C-3 of maytansinoid, an antibody To the maytansinoids, wherein the thiol- or disulfide functional group of the acyl group of the acylated amino acid side chain is on a carbon atom with one or two substituents, and these substituents are CH ₃ , C ₂ H ₅ , straight chain alkyl or alkenyl having 1 to 10 carbon atoms, branched or cyclic alkyl or alkenyl having 3 to 10 carbon atoms, phenyl, substituted phenyl, or heterocyclic aromatic or hetero A cyclic group, and one of the substituents may be H, and the acyl group is less between the carbonyl functional group and the sulfur atom. Kutomo with three linear carbon atoms in length.

Wherein A, B and D are each independently a cycloalkyl or cycloalkenyl having 3 to 10 carbon atoms, a simple or substituted aryl or a heterocyclic aromatic or heterocyclic group. Yes;
R ₃ , R ₄ , R ₅ , R ₆ , R ₇ , R ₈ , R ₉ , R ₁₀ , R ₁₁ and R ₁₂ are each independently H, CH ₃ , C ₂ H ₅ , 1 to 10 A linear alkyl or alkenyl having 3 carbon atoms, a branched or cyclic alkyl or alkenyl having 3 to 10 carbon atoms, phenyl, substituted phenyl, or a heterocyclic aromatic or heterocyclic group;
l, m, n, o, p, q, r, s, t and u are each independently an integer of 0 or 1 to 5, provided that l, m, n, o, p, q, r, At least two of s, t and u are not zero in any case.

Claims (97)

SEQ ID NO: amino acid sequence selected from the group consisting of 1-6:

An antibody or epitope-binding fragment thereof comprising at least one complementarity determining region having An amino acid comprising at least one heavy chain variable region or fragment thereof and at least one light chain variable region or fragment thereof, wherein at least one heavy chain variable region or fragment thereof is represented by SEQ ID NO: 1 to 3, respectively. Array:

An amino acid sequence comprising at least one light chain variable region or a fragment thereof each represented by SEQ ID NOs: 4-6:

An antibody or epitope-binding fragment thereof comprising three sequential sequence complementarity determining regions having The amino acid sequence in which the light chain variable region or a fragment thereof is represented by SEQ ID NO: 7 or SEQ ID NO: 8:

3. The antibody or epitope-binding fragment thereof according to claim 2, having at least 90% sequence identity to.   4. The antibody or epitope-binding fragment thereof according to claim 3, wherein the light chain variable region or fragment thereof has at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 7 or SEQ ID NO: 8.   The antibody or epitope-binding fragment thereof according to claim 3, wherein the light chain variable region or a fragment thereof has an amino acid sequence of SEQ ID NO: 7 or SEQ ID NO: 8. An amino acid sequence wherein the heavy chain variable region or fragment thereof is selected from the group consisting of SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 11.

3. The antibody or epitope-binding fragment thereof according to claim 2, having at least 90% sequence identity to.   The antibody according to claim 6, wherein the heavy chain variable region or a fragment thereof has at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 9, SEQ ID NO: 10 or SEQ ID NO: 11. Its epitope binding fragment.   The antibody or epitope-binding fragment thereof according to claim 6, wherein the heavy chain variable region or a fragment thereof has an amino acid sequence of SEQ ID NO: 9, SEQ ID NO: 10 or SEQ ID NO: 11.   A polynucleotide encoding the antibody or epitope-binding fragment thereof according to any one of claims 1, 2, 3 or 6.   A polynucleotide encoding the L chain or H chain of the antibody or epitope-binding fragment thereof according to any one of claims 1, 2, 3 or 6.   A vector comprising the polynucleotide according to claim 9.   A vector comprising the polynucleotide according to claim 10.   12. The vector of claim 11, wherein the vector is an expression vector that expresses the antibody or epitope-binding fragment thereof.   13. The vector according to claim 12, wherein the vector is an expression vector that expresses the antibody or epitope-binding fragment thereof. A humanized rodent antibody or epitope-binding fragment thereof that binds to a CA6 glycotope produced by a resurfacing method comprising:
(A) A position alignment was created from the relative accessibility distribution obtained by the x-ray crystallographic structure of the rodent and human antibody heavy and light chain variable region pools, and the heavy and light chain variable region framework Determining a set of positions exposed on the surface, wherein the alignment positions of all rodent and human variable regions are at least about 98% identical;
(B) For a rodent antibody or epitope-binding fragment thereof, using a set of positions exposed on the surface of the heavy and light chain variable region framework determined in step (a), the heavy and light chain variable regions Determine a set of amino acid residues exposed on the framework surface;
(C) From the human antibody amino acid sequence, the H chain and L chain variable regions having the closest identity to the set of amino acid residues exposed on the surface of the H chain and L chain variable region framework determined in step (b) Identify a set of amino acid residues exposed on the framework surface;
(D) In the variable region framework amino acid sequence of the rodent antibody or epitope-binding fragment thereof, a set of amino acid residues exposed on the surface of the heavy chain and light chain variable region frameworks determined in step (b), Substituting with a set of amino acid residues exposed on the H and L chain variable region framework surfaces identified in step (c);
(E) Build a three-dimensional model of the variable region of the rodent antibody or epitope-binding fragment thereof, and the variable region of the rodent antibody or epitope-binding fragment thereof obtained by the substitution specified in step (d). ;
(F) Comparing the three-dimensional model constructed in step (e), and from the set determined in step (b) or (c), any of the complementarity determining regions of rodent antibodies or epitope-binding fragments thereof Identify amino acid residues within 5 cm of any atom of the residue;
(G) replacing any of the residues identified in step (f) from human amino acid residues to the original rodent amino acid residues, thereby humanizing the amino acid residues exposed on the surface; Determine the pair;
(H) A set of amino acid residues exposed on the surface of the rodent antibody variable region framework determined in step (b) was exposed on the surface of the variable region framework for humanization determined in step (g). Making a set of amino acid residues; and (i) making a humanized rodent antibody or epitope-binding fragment thereof that binds to the CA6 glycotope.   16. The humanized rodent antibody or epitope-binding fragment thereof according to claim 15, wherein the humanized rodent antibody or epitope-binding fragment thereof is an epitope-binding fragment. Epitope binding fragments are Fab fragments, Fab ′ fragments, F (ab ′) ₂ fragments, Fd fragments, single chain Fv (scFv) fragments, single chain antibodies, disulfide bond Fv (sdFv) fragments, and _VL or V 17. The humanized rodent antibody or epitope-binding fragment thereof according to claim 16, selected from the group consisting of fragments comprising any of the _H domains.   The humanized rodent antibody or epitope-binding fragment thereof according to claim 15 or 16, wherein the amino acid residue exposed on the surface is a residue having a solvent accessibility of more than 30%. SEQ ID NO: amino acid sequence selected from the group consisting of 1-6:

16. The humanized rodent antibody or epitope-binding fragment thereof according to claim 15, comprising at least one complementarity determining region having An amino acid comprising at least one heavy chain variable region or fragment thereof and at least one light chain variable region or fragment thereof, wherein at least one heavy chain variable region or fragment thereof is represented by SEQ ID NO: 1 to 3, respectively. Array:

An amino acid sequence comprising at least one light chain variable region or a fragment thereof each represented by SEQ ID NOs: 4-6:

16. The humanized rodent antibody or epitope-binding fragment thereof according to claim 15, comprising three sequential sequence complementarity determining regions having: Amino acid sequence of SEQ ID NO: 7 or SEQ ID NO: 8:

16. The humanized rodent antibody or epitope-binding fragment thereof according to claim 15, comprising a light chain variable region having sex. An amino acid sequence selected from the group consisting of SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 11.

The humanized rodent antibody or epitope-binding fragment thereof according to claim 15, comprising a heavy chain variable region having: An improved antibody or epitope-binding fragment thereof that binds to a CA6 glycotope-expressing cell, the improved antibody or epitope-binding fragment thereof produced by:
(A) SEQ ID NO: 1 to 11:

Preparing a DNA polynucleotide encoding an antibody or epitope-binding fragment thereof comprising at least one sequence selected from the group consisting of:
(B) introducing at least one nucleotide mutation, deletion or insertion into the DNA polynucleotide of (a) so as to change at least one residue of the amino acid sequence encoded by the DNA polynucleotide;
(C) expressing an antibody or antibody fragment encoded by the DNA polynucleotide of (b); and (d) screening the expressed antibody or antibody fragment for improved binding affinity to CA6 glycotope expressing cells, thereby Improved antibodies or epitope binding fragments thereof are obtained.   Mutation, deletion or insertion of at least one nucleotide from oligonucleotide-mediated site-directed mutagenesis, cassette mutagenesis, error-prone PCR, DNA shuffling, and use of E. coli mutator strains 24. The improved antibody or epitope-binding fragment thereof according to claim 23, carried out by a method selected from the group consisting of:   A cytotoxic conjugate comprising a cell binding agent and a cytotoxic agent, wherein the cell binding agent binds to a CA6 glycotope.   26. The cytotoxic conjugate of claim 25, wherein the cell binding agent and cytotoxic agent are covalently bound.   26. The cytotoxic conjugate of claim 25, wherein the cell binding agent and cytotoxic agent are covalently linked by a PEG linking group.   26. The cytotoxic conjugate of claim 25, wherein the cell binding agent and cytotoxic agent are covalently linked by a thiol or disulfide functional group of the cytotoxic agent.   Cell binding agent selected from the group consisting of polyclonal antibodies, monoclonal antibodies, antibody fragments, humanized or resurfaced antibodies, functional equivalents of antibodies, improved antibodies, interferons, lymphokines, hormones, growth factors, transferrin and vitamins 26. The cytotoxic conjugate of claim 25, wherein the cytotoxic conjugate is a Antibody fragments include Fab fragments, Fab ′ fragments, F (ab ′) ₂ fragments, Fd fragments, single chain Fv (scFv) fragments, single chain antibodies, disulfide bond Fv (sdFv) fragments, and VL or VH domains 30. The cytotoxic conjugate of claim 29, selected from the group consisting of fragments comprising any of   26. The cytotoxic conjugate of claim 25, wherein the cell binding agent is an anti-CA6 monoclonal antibody or an epitope binding fragment thereof.   26. The cytotoxic conjugate of claim 25, wherein the cell binding agent is murine anti-CA6 monoclonal antibody DS6 or an epitope-binding fragment thereof.   26. The cytotoxic conjugate of claim 25, wherein the cell binding agent is a humanized version of murine anti-CA6 monoclonal antibody DS6 or an epitope-binding fragment thereof.   26. The cytotoxic conjugate of claim 25, wherein the cytotoxic agent is a member selected from the group consisting of maytansinoid compounds, taxoid compounds, CC-1065 compounds, dolastatin compounds, daunorubicin compounds, and doxorubicin compounds. The cytotoxic substance is maytansine DM1: of formula (I)

35. The cytotoxic conjugate of claim 34, wherein The cytotoxic substance is maytansine DM4 of formula (II):

35. The cytotoxic conjugate of claim 34, wherein The cytotoxic substance is maytansine of formula (III):

[Where:
Y '

Wherein R ₁ and R ₂ are each independently CH ₃ , C ₂ H ₅ , a linear alkyl or alkenyl having 1 to 10 carbon atoms, and a moiety having 3 to 10 carbon atoms. A branched or cyclic alkyl or alkenyl, phenyl, substituted phenyl, or heterocyclic aromatic or heterocycloalkyl group, and R ₂ may be H;
A, B, D are cycloalkyl or cycloalkenyl having 3 to 10 carbon atoms, simple or substituted aryl or heterocyclic aromatic or heterocycloalkyl group;
R ₃ , R ₄ , R ₅ , R ₆ , R ₇ , R ₈ , R ₉ , R ₁₁ and R ₁₂ are each independently H, CH ₃ , C ₂ H ₅ , 1-10 carbon atoms. A straight-chain alkyl or alkenyl having 3, a branched or cyclic alkyl or alkenyl having 3 to 10 carbon atoms, phenyl, substituted phenyl, or a heterocyclic aromatic or heterocycloalkyl group;
l, m, n, o, p, q, r, s and t are each independently an integer of 0 or 1 to 5, provided that l, m, n, o, p, q, r, s and at least two of t are not zero in any case;
Z is H, SR or —COR, wherein R is a straight chain alkyl or alkenyl having 1 to 10 carbon atoms, a branched or cyclic alkyl or alkenyl having 3 to 10 carbon atoms, Or a simple or substituted aryl or heteroaromatic or heterocycloalkyl group]. 38. The cytotoxic conjugate of claim 37, wherein R ₁ is H, R ₂ is methyl, and Z is H. R ₁ and R ₂ are methyl, Z is H, the cytotoxic conjugate according to claim 37. R ₁ is H, _{R 2} is methyl, Z is -SCH _3, the cytotoxic conjugate according to claim 37. R ₁ and _{R 2} are methyl, Z is -SCH _3, the cytotoxic conjugate according to claim 37. Maytansine wherein the cytotoxic agent is selected from the group consisting of formulas (IV-L), (IV-D) and (IV-D, L):

[Where:
Y is

Wherein R ₁ and R ₂ are each independently CH ₃ , C ₂ H ₅ , a linear alkyl or alkenyl having 1 to 10 carbon atoms, and a moiety having 3 to 10 carbon atoms. A branched or cyclic alkyl or alkenyl, phenyl, substituted phenyl, or heterocyclic aromatic or heterocycloalkyl group, and R ₂ may be H;
R ₃ , R ₄ , R ₅ , R ₆ , R ₇ and R ₈ are each independently H, CH ₃ , C ₂ H ₅ , linear alkyl or alkenyl having 1 to 10 carbon atoms, 3 A branched or cyclic alkyl or alkenyl having 10 to 10 carbon atoms, phenyl, substituted phenyl, or a heterocyclic aromatic or heterocycloalkyl group;
l, m and n are each independently an integer of 1 to 5, and n may be 0;
Z is H, SR or —COR, wherein R is a linear or branched alkyl or alkenyl having 1 to 10 carbon atoms, a cyclic alkyl or alkenyl having 3 to 10 carbon atoms, Or a simple or substituted aryl or heterocyclic aromatic or heterocycloalkyl group;
35. May represents a maytansinoid having a C-3 side chain, C-14 hydroxymethyl, C-15 hydroxy, or C-20 desmethyl] . R ₁ is H, R ₂ is methyl, R ₅ , R ₆ , R ₇ and R ₈ are each H, l and m are each 1, n is 0, Z is H 43. The cytotoxic conjugate of claim 42, wherein 43. R ₁ and R ₂ are methyl, R ₅ , R ₆ , R ₇ and R ₈ are each H, l and m are 1, n is 0 and Z is H A cytotoxic conjugate according to claim 1. R ₁ is H, R ₂ is methyl, R ₅ , R ₆ , R ₇ and R ₈ are each H, l and m are each 1, n is 0, and Z is —SCH 43. The cytotoxic conjugate of claim 42, which is ₃ . R ₁ and R ₂ are methyl, R ₅ , R ₆ , R ₇ and R ₈ are each H, l and m are 1, n is 0, and Z is —SCH _3. Item 43. The cytotoxic conjugate according to Item 42.   The cytotoxic conjugate according to claim 42, wherein the cytotoxic substance is represented by formula (IV-L). The cytotoxic substance is maytansine of formula (V):

[Where:
Y is

Wherein R ₁ and R ₂ are each independently CH ₃ , C ₂ H ₅ , a linear alkyl or alkenyl having 1 to 10 carbon atoms, and a moiety having 3 to 10 carbon atoms. A branched or cyclic alkyl or alkenyl, phenyl, substituted phenyl, or heterocyclic aromatic or heterocycloalkyl group, and R ₂ may be H;
R ₃ , R ₄ , R ₅ , R ₆ , R ₇ and R ₈ are each independently H, CH ₃ , C ₂ H ₅ , linear alkyl or alkenyl having 1 to 10 carbon atoms, 3 A branched or cyclic alkyl or alkenyl having 10 to 10 carbon atoms, phenyl, substituted phenyl, or a heterocyclic aromatic or heterocycloalkyl group;
l, m and n are each independently an integer of 1 to 5, and n may be 0;
Z is H, SR or —COR, wherein R is a straight chain alkyl or alkenyl having 1 to 10 carbon atoms, a branched or cyclic alkyl or alkenyl having 3 to 10 carbon atoms, Or a simple or substituted aryl or heteroaromatic or heterocycloalkyl group].   49. In claim 48, R1 is H, R2 is methyl, R5, R6, R7 and R8 are each H; l and m are each 1; n is 0; and Z is H The cytotoxic conjugate as described. 49. R ₁ and R ₂ are methyl; R ₅ , R ₆ , R ₇ and R ₈ are each H; l and m are 1; n is 0; A cytotoxic conjugate according to claim 1. R ₁ is H, R ₂ is methyl, R ₅ , R ₆ , R ₇ and R ₈ are each H, l and m are each 1, n is 0, and Z is —SCH 49. The cytotoxic conjugate of claim 48, which is ₃ . R ₁ and R ₂ are methyl, R ₅ , R ₆ , R ₇ and R ₈ are each H, l and m are 1, n is 0, and Z is —SCH _3. Item 49. The cytotoxic conjugate according to Item 48. Maytansine wherein the cytotoxic agent is selected from the group consisting of formulas (VI-L), (VI-D) and (VI-D, L):

[Where:
Y ₂ is,

Wherein R ₁ and R ₂ are each independently CH ₃ , C ₂ H ₅ , a linear alkyl or alkenyl having 1 to 10 carbon atoms, and a moiety having 3 to 10 carbon atoms. A branched or cyclic alkyl or alkenyl, phenyl, substituted phenyl, or heterocyclic aromatic or heterocycloalkyl group, and R ₂ may be H;
R ₃ , R ₄ , R ₅ , R ₆ , R ₇ and R ₈ are each independently H, CH ₃ , C ₂ H ₅ , linear cyclic alkyl or alkenyl having 1 to 10 carbon atoms. A branched or cyclic alkyl or alkenyl, phenyl, substituted phenyl, or heterocyclic aromatic or heterocycloalkyl group having 3 to 10 carbon atoms;
l, m and n are each independently an integer of 1 to 5, and n may be 0;
Z ₂ is SR or COR, where R is a linear alkyl or alkenyl having 1 to 10 carbon atoms, a branched or cyclic alkyl or alkenyl having 3 to 10 carbon atoms, or simply Or a substituted aryl or heterocyclic aromatic or heterocycloalkyl group;
35. The cytotoxic conjugate of claim 34, wherein May is a maytansinoid. The cytotoxic substance is maytansine of formula (VII):

[Where:
Y ₂ '

Wherein R ₁ and R ₂ are each independently CH ₃ , C ₂ H ₅ , straight chain branched alkyl or alkenyl having 1 to 10 carbon atoms, and 3 to 10 carbon atoms. A cyclic alkyl or alkenyl having a phenyl, substituted phenyl, or a heterocyclic aromatic or heterocycloalkyl group, and R ₂ may be H;
A, B and D are each independently a cycloalkyl or cycloalkenyl, simple or substituted aryl or heteroaromatic or heterocycloalkyl group having from 3 to 10 carbon atoms;
R ₃ , R ₄ , R ₅ , R ₆ , R ₇ , R ₈ , R ₉ , R ₁₁ and R ₁₂ are each independently H, CH ₃ , C ₂ H ₅ , 1-10 carbon atoms. A straight-chain alkyl or alkenyl having 3, a branched or cyclic alkyl or alkenyl having 3 to 10 carbon atoms, phenyl, substituted phenyl, or a heterocyclic aromatic or heterocycloalkyl group;
l, m, n, o, p, q, r, s and t are each independently an integer of 0 or 1 to 5, provided that l, m, n, o, p, q, r, s and at least two of t are not zero in any case;
Z ₂ is SR or —COR, wherein R is a linear alkyl or alkenyl having 1 to 10 carbon atoms, a branched or cyclic alkyl or alkenyl having 3 to 10 carbon atoms, or 35. A cytotoxic conjugate according to claim 34, which is a simple or substituted aryl or heterocyclic aromatic or heterocycloalkyl group.   55. The cytotoxic conjugate of claim 54, wherein R1 is H and R2 is methyl. The cytotoxic substance is maytansine of formula (VIII):

[Where:
Y ₁ '

Wherein A, B and D are each independently a cycloalkyl or cycloalkenyl having 3 to 10 carbon atoms, a simple or substituted aryl or heterocyclic aromatic or heterocycloalkyl group. Yes;
R ₃ , R ₄ , R ₅ , R ₆ , R ₇ , R ₈ , R ₉ , R ₁₁ and R ₁₂ are each independently H, CH ₃ , C ₂ H ₅ , 1-10 carbon atoms. A straight-chain alkyl or alkenyl having 3, a branched or cyclic alkyl or alkenyl having 3 to 10 carbon atoms, phenyl, substituted phenyl, or a heterocyclic aromatic or heterocycloalkyl group;
l, m, n, o, p, q, r, s and t are each independently an integer of 0 or 1 to 5, provided that l, m, n, o, p, q, r, s and 35. The cytotoxic conjugate of claim 34, wherein at least two of t are not non-zero in any case. R ₁ is H, R ₂ is methyl, the cytotoxic conjugate according to claim 56. R ₁ and R ₂ are methyl, cytotoxic conjugate according to claim 56. A maytansine wherein the cytotoxic agent is selected from the group consisting of formulas (IX-L), (IX-D) and (IX-D, L):

[Where:
Y ₁ is,

Wherein R ₁ and R ₂ are each independently CH ₃ , C ₂ H ₅ , a linear alkyl or alkenyl having 1 to 10 carbon atoms, and a moiety having 3 to 10 carbon atoms. A branched or cyclic alkyl or alkenyl, phenyl, substituted phenyl, or heterocyclic aromatic or heterocycloalkyl group, and R ₂ may be H;
R ₃ , R ₄ , R ₅ , R ₆ , R ₇ and R ₈ are each independently H, CH ₃ , C ₂ H ₅ , linear alkyl or alkenyl having 1 to 10 carbon atoms, 3 A branched or cyclic alkyl or alkenyl having 10 to 10 carbon atoms, phenyl, substituted phenyl, or a heterocyclic aromatic or heterocycloalkyl group;
l, m and n are each independently an integer of 1 to 5, and n may be 0;
35. May represents a maytansinoid having a C-3 side chain, C-14 hydroxymethyl, C-15 hydroxy, or C-20 desmethyl] . R ₁ is H, or _{R 2} is methyl, or _{R 1} and _{R 2} are methyl, cytotoxic conjugate according to claim 59. 60. In claim 59, wherein R ₁ is H, R ₂ is methyl, R ₅ , R ₆ , R ₇ and R ₈ are each H; l and m are each 1; and n is 0 The cytotoxic conjugate as described. R ₁ and _{R 2} are _{methyl; R 5, R 6, R} 7 and _{R 8} are each H; l and m is 1; n is 0, cytotoxic Gongju according to claim 59 Gate.   61. The cytotoxic conjugate according to claim 60, wherein the cytotoxic substance is represented by formula (IX-L).   62. The cytotoxic conjugate according to claim 61, wherein the cytotoxic substance is represented by formula (IX-L).   63. The cytotoxic conjugate according to claim 62, wherein the cytotoxic substance is represented by formula (IX-L). The cytotoxic substance is maytansine of formula (X):

[Where:
Y ₁ is,

Wherein R ₁ and R ₂ are each independently CH ₃ , C ₂ H ₅ , a linear alkyl or alkenyl having 1 to 10 carbon atoms, and a moiety having 3 to 10 carbon atoms. A branched or cyclic alkyl or alkenyl, phenyl, substituted phenyl, or heterocyclic aromatic or heterocycloalkyl group, and R ₂ may be H;
R ₃ , R ₄ , R ₅ , R ₆ , R ₇ and R ₈ are each independently H, CH ₃ , C ₂ H ₅ , linear alkyl or alkenyl having 1 to 10 carbon atoms, 3 A branched or cyclic alkyl or alkenyl having 10 to 10 carbon atoms, phenyl, substituted phenyl, or a heterocyclic aromatic or heterocycloalkyl group;
l, m and n are each independently an integer of 1 to 5, and n may be 0;
35. May represents a maytansinoid having a C-3 side chain, C-14 hydroxymethyl, C-15 hydroxy, or C-20 desmethyl] . 67. In claim 66, R ₁ is H, R ₂ is methyl, R ₅ , R ₆ , R ₇ and R ₈ are each H, l and m are each 1 and n is 0. The cytotoxic conjugate as described. R ₁ and _{R 2} are _{methyl; R 5, R 6, R} 7 and _{R 8} are each H; l and m is 1; n is 0, cytotoxic Gongju according to claim 66 Gate.   26. The cytotoxic conjugate of claim 25, wherein the cell binding agent is a humanized version of murine anti-CA6 monoclonal antibody DS6 or an epitope binding fragment thereof and the cytotoxic agent is DM1 or DM4. The cytotoxic substance is a taxane of formula (II):

35. The cytotoxic conjugate of claim 34, wherein   26. A method of inhibiting the growth of a CA6 glycotope expressing cell, comprising contacting the CA6 glycotope expressing cell with the cytotoxic conjugate of claim 25.   72. The method of claim 71 for inhibiting the growth of CA6 glycotope expressing cells, wherein the cytotoxic conjugate is an anti-CA6 monoclonal antibody or epitope-binding fragment thereof as a cell binding agent and DM1 or DM4 as a cytotoxic agent. Including methods.   72. The method of claim 71 for inhibiting proliferation of CA6 glycotope expressing cells, wherein the cytotoxic conjugate comprises an anti-CA6 monoclonal antibody or epitope-binding fragment thereof as a cell binding agent and taxanes as a cytotoxic agent. Including methods.   72. The method of claim 71 for inhibiting proliferation of a CA6 glycotope expressing cell, wherein the cytotoxic conjugate is a humanized version of murine antibody DS6 as a cell binding agent or epitope binding fragment thereof and as a cytotoxic agent. A method comprising DM1 or DM4.   72. The method of claim 71 for inhibiting proliferation of a CA6 glycotope expressing cell, wherein the cytotoxic conjugate is a humanized version of murine antibody DS6 as a cell binding agent or epitope binding fragment thereof and as a cytotoxic agent. A method comprising taxanes.   72. The method of claim 71 for inhibiting the growth of a CA6 glycotope expressing cell, wherein the cell is killed by inhibiting the growth.   72. The method of claim 71 for inhibiting the growth of CA6 glycotope expressing cells, wherein said method is performed in vivo, in vitro or ex vivo.   26. A therapeutic composition comprising the cytotoxic conjugate of claim 25 and a pharmaceutically acceptable carrier or excipient.   79. The therapeutic composition of claim 78, wherein the cytotoxic conjugate comprises a humanized version of murine antibody DS6 or an epitope-binding fragment thereof as a cell binding agent and DM1 or DM4 as a cytotoxic agent. 80. The therapeutic composition of claim 78, wherein the cytotoxic conjugate comprises a humanized version of murine antibody DS6 or an epitope-binding fragment thereof as a cell binding agent and taxanes as a cytotoxic agent.   79. A method of treating a subject with cancer, comprising administering to the subject with cancer a therapeutically effective amount of the therapeutic composition of claim 78, thereby treating the subject with cancer.   92. A method of treating a subject with cancer according to claim 81, wherein the cytotoxic conjugate comprises a humanized version of murine antibody DS6 or an epitope-binding fragment thereof as a cell binding agent and DM1 or DM4 as a cytotoxic agent.   82. A method of treating a subject with cancer according to claim 81, wherein the cytotoxic conjugate comprises a humanized version of murine antibody DS6 or an epitope-binding fragment thereof as a cell binding agent and taxanes as a cytotoxic agent.   82. The method for treating a subject with cancer according to claim 81, wherein the cancer is a cancer that expresses or overexpresses a CA6 glycotope.   The cancer is from the group consisting of serous ovarian cancer, endometrial ovarian cancer, cervical neoplasm, endometrial neoplasm, vulvar neoplasm, breast cancer, pancreatic tumor, and urothelial tumor 82. A method of treating a subject with cancer according to claim 81, wherein the subject is selected. 26. A kit comprising the cytotoxic conjugate of claim 25, further comprising: a) a compartment containing the cytotoxic conjugate; and b) instructions for using the kit.   87. The kit of claim 86, wherein the cytotoxic conjugate comprises a humanized version of murine antibody DS6 or an epitope-binding fragment thereof as a cell binding agent and DM1 or DM4 as a cytotoxic agent.   87. The kit of claim 86, wherein the cytotoxic conjugate comprises a humanized version of murine antibody DS6 or an epitope-binding fragment thereof as a cell binding agent and taxanes as a cytotoxic agent.   90. The kit of claim 86, wherein the instructions comprise instructions for treating a subject with cancer.   90. The kit of claim 89, wherein the cancer is a cancer that expresses or overexpresses the CA6 glycotope.   The cancer is from the group consisting of serous ovarian cancer, endometrial ovarian cancer, cervical neoplasm, endometrial neoplasm, vulvar neoplasm, breast cancer, pancreatic tumor, and urothelial tumor The kit of claim 90, wherein the kit is selected. 79. A kit comprising the therapeutic composition of claim 78, further comprising: a) a compartment containing the therapeutic composition; and b) instructions for using the kit. 94. The kit of claim 92, wherein the cytotoxic conjugate comprises a humanized version of murine antibody DS6 or an epitope-binding fragment thereof as a cell binding agent and DM1 or DM4 as a cytotoxic agent.   94. The kit of claim 92, wherein the cytotoxic conjugate comprises a humanized version of murine antibody DS6 or an epitope-binding fragment thereof as a cell binding agent and taxanes as a cytotoxic agent.   94. The kit of claim 92, wherein the instructions comprise instructions for treating a subject with cancer.   96. The kit of claim 95, wherein the cancer is a cancer that expresses or overexpresses a CA6 glycotope.   The cancer is from the group consisting of serous ovarian cancer, endometrial ovarian cancer, cervical neoplasm, endometrial neoplasm, vulvar neoplasm, breast cancer, pancreatic tumor, and urothelial tumor 99. The kit of claim 96, wherein the kit is selected.

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